Modified fgf-21 polypeptides and their uses

ABSTRACT

Modified FGF-21 polypeptides and uses thereof are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 16/443,226filed Jun. 17, 2019, now U.S. Pat. No. 10,961,291, which is a divisionalof U.S. application Ser. No. 15/953,091 filed Apr. 13, 2018, now U.S.Pat. No. 10,377,805, which is a divisional of U.S. application Ser. No.15/292,700, filed Oct. 13, 2016, now U.S. Pat. No. 9,975,936, which is adivisional of U.S. patent application Ser. No. 14/680,543, filed Apr. 7,2015, now U.S. Pat. No. 9,517,273, which is a divisional of U.S. patentapplication Ser. No. 13/732,522, filed Jan. 2, 2013, now U.S. Pat. No.9,079,971, which is a divisional of U.S. patent application Ser. No.13/051,953, filed Mar. 18, 2011, now U.S. Pat. No. 8,383,365, which is adivisional of U.S. patent application Ser. No. 12/051,830, filed Mar.19, 2008, now U.S. Pat. No. 8,012,931, which claims the benefit of U.S.Provisional Application No. 60/988,060, filed Nov. 14, 2007, and alsoclaims the benefit of U.S. Provisional Application No. 60/921,297, filedMar. 30, 2007, each of which is hereby incorporated by reference in itsentirety.

SEQUENCE LISTING

This application includes a sequence listing which has been submittedvia EFS-Web in a file named “1143270o001010.txt” created on Feb. 2, 2021and having a size of 87,144 bytes, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to FGF-21 polypeptides optionally modified withat least one non-naturally-encoded amino acid.

BACKGROUND OF THE INVENTION

Fibroblast growth factors are large polypeptides widely expressed indeveloping and adult tissues (Baird et al., Cancer Cells, 3:239-243,1991) and play crucial roles in multiple physiological functionsincluding angiogenesis, mitogenesis, pattern formation, cellulardifferentiation, metabolic regulation and repair of tissue injury(McKeehan et al., Prog. Nucleic Acid Res. Mol. Biol. 59:135-176, 1998;Burgess, W. H. et al., Annu. Rev. Biochem. 58:575-606 (1989). Theprototypic fibroblast growth factors (FGFs), FGF-1 and FGF-2, wereoriginally isolated from brain and pituitary as mitogens forfibroblasts. FGF-3 was identified to be a common target for activationby the mouse mammary tumor virus (Dickson et al., Ann. N.Y. Acad. Sci.638:18-26 (1991); FGF-4 to FGF-6 were identified as oncogene products(Yoshida et al., Ann. NY Acad. Sci. 638:27-37 (1991); Goldfarb et al.,Ann. NY Acad. Sci 638:38-52 (1991); Coulier et al., Ann. NY Acad. Sci.638:53-61 (1991)). FGF-10 was identified from rat lung by homology-basedpolymerase chain reaction (PCR) (Yamasaki et al., J. Biol. Chem.271:15918-15921 (1996)). FGF-11 to FGF-14 (FGF homologous factors (FHFs)1 to 4) were identified from human retina by a combination of randomcDNA sequencing, database searches and homology-based PCR (Smallwood etal., Proc. Natl. Acad. Sci. USA 93:9850-9857 (1996)). FGF-15 wasidentified as a downstream target of a chimeric homeodomain oncoprotein(McWhirter et al., Development 124:3221-3232 (1997)). FGF-16, FGF-17,and FGF-18 were identified from rat heart and embryos by homology-basedPCR, respectively (Miyake et al., Biochem. Biophys. Res. Commun.243:148-152 (1998); Hoshikawa et al. Biochem. Biophys. Res. Commun.244:187-191 (1998); Ohbayashi et al., J. Biol. Chem. 273:18161-18164(1998)). FGF-19 was identified from human fetal brain by database search(Nishimura et al., Biochim. Biophys. Acta 1444:148-151 (1999)). Theyhave a conserved ˜120-amino acid residue core with ˜30 to 60% amino acididentity.

Animal models, overexpression, and analysis of naturally occurringmutations implicate fibroblast growth factors and their receptors in awide range of diseases (e.g. Wilkie et al., Current Biology, (1995)5:500-507; Pugh-Humphreys et al, In: The Cytokine Handbook, A. Thomsoned, 2nd edition, Academic Press, Harcourt Brace & co. publishers,London, pp 525-566) suggesting that regulation of activity could be usedfor treatment. For example, inhibition of fibroblast growth factor-2 bythe compound Suramin prevents neovascularisation and tumor growth inmice (Pesenti et al., British Journal of Cancer, 66:367-372). Fibroblastgrowth factors also function in angiogenesis (Lyons, M. K., et al.,Brain Res. (1991) 558:315-320), wound healing (Uhl, E., et al., Br. J.Surg. (1993) 80:977-980, 1993), astrogliosis, glial cell proliferationand differentiation (Biagini, G. et al., Neurochem. Int. (1994)25:17-24), cerebral vasodilation (Tanaka, R. et al., Stroke (1995)26:2154-2159), and neurotrophic/neuromodulatory processes.

Fibroblast growth factor also has multiple positive effects includingblood flow and protection from calcium toxicity to improve outcome incerebral ischemia (Mattson, M. P. et al., Semin. Neurosci. (1993)5:295-307; Doetrocj. W. D. et al., J. Neurotrauma (1996) 13:309-316).Basic FGF treatment promotes neoangiogenesis in ischemic myocardium(Schumacher et al., Circulation (1998) 97: 645-650). Basic FGF enhancesfunctional recovery and promotes neuronal sprouting following focalcerebral infarct (Kawamata et al., Proc.Natl. Acad. Sci. (1997) 94(15):8179-84). According to the published literature, the FGF familyconsists of at least twenty-two members (Reuss et al., Cell Tissue Res.313:139-157 (2003)).

Fibroblast growth factor 21 (FGF-21) has been reported to bepreferentially expressed in the liver (Nishimura et al., Biochimica etBiophysica Acta, 1492:203-206 (2000); WO 01/36640; and WO 01/18172,which are incorporated by reference herein) and described as a treatmentfor ischemic vascular disease, wound healing, and diseases associatedwith loss of pulmonary, bronchia or alvelor cells or function andnumerous other disorders. FGF-21 is expressed primarily in liver,kidney, and muscle tissue (see Example 2 of US Patent Publication No.20040259780 which is incorporated by reference herein in its entirety).The FGF-21 gene is composed of 3 exons and is located on chromosome 19.Unlike other FGFs, FGF-21 does not have proliferative and tumorigeniceffects (Genome Biol. 2001; 2(3):REVIEWS3005).

US Patent Publication No. 20010012628, which is incorporated byreference in its entirety, describes a nucleotide and protein sequencefor human FGF-21 (see SEQ ID NO: 1 and 2, respectively of US PatentPublication No. 20010012628). SEQ ID NO: 2 in the above-mentionedpublication, referred to sbgFGF-19, is 209 amino acids in length andcontains a 28 amino acid leader sequence at the N terminus. The humanFGF-21 sequence presented as SEQ ID NO: 3 herein is the same sequence asSEQ ID NO: 2 of US Patent Publication No. 20010012628. This sequence hasa single nucleotide polymorphism (SNP) with proline (P) at position 174,hereinafter referred to as the “209 amino acid P-form of FGF-21.”

U.S. Pat. No. 6,716,626, which is incorporated by reference herein inits entirety, discuss human FGF-21 and homologous proteins in othermammals, particularly mice and rats. Mouse FGF shown as SEQ ID NO: 1 ofU.S. Pat. No. 6,716,626 was highly expressed in liver and expressed inthe testis and thymus, and it was suggested that human FGF-21 may play arole in development of and recovery from liver disease and/or disordersof testicular function or function of cells derived from the thymus. SEQID NO: 4 of U.S. Pat. No. 6,716,626 is 209 amino acids in length andcontains a 28 amino acid leader sequence at the N terminus. The humanFGF-21 sequence presented as SEQ ID NO: 6 herein is the same sequence asSEQ ID NO: 4 of U.S. Pat. No. 6,716,626. This sequence has a singlenucleotide polymorphism (SNP) with leucine (L) at position 174,hereinafter referred to as the “209 amino acid L-form of FGF-21.”

U.S. Patent Publication No. 20040259780, which is incorporated byreference herein in its entirety, discuss human FGF-21 and present asequence that is 208 amino acids in length (SEQ ID NO: 2 of U.S. PatentPublication No. 20040259780) and contains a 27 amino acid leadersequence at the N terminus. The human FGF-21 sequence presented as SEQID NO: 7 herein is the same sequence as SEQ ID NO: 2 of U.S. PatentPublication No. 20040259780. This sequence has a single nucleotidepolymorphism (SNP) with leucine (L) at position 173, herein afterreferred to as the “208 amino acid L-form of FGF-21.”

FGF-21 has been shown to stimulate glucose-uptake in mouse 3T3-L1adipocytes in the presence and absence of insulin, and to decrease fedand fasting blood glucose, triglycerides, and glucagon levels in ob/oband db/db mice and 8 week old ZDF rats in a dose-dependent manner, thus,providing the basis for the use of FGF-21 as a therapy for treatingdiabetes and obesity (WO 03/011213, which is incorporated by referenceherein and Kharitonenkov et al. J Clin Invest. 2005 June;115(6):1627-35). Kharitonenkov et al. J Clin Invest. 2005 June;115(6):1627-35 also showed that transgenic mice expressing human FGF-21are hypoglycemic, sensitive to insulin, and resistant to diet-inducedobesity. Kharitonenkov et al. Endocrinology (in press) also show thatFGF-21 lowered glucose, triglycerides, insulin, and glucagons indiabetic Rhesus monkeys.

In addition, FGF-21 has been shown to be effective in reducing themortality and morbidity of critically ill patients (WO 03/059270, whichis incorporated by reference herein). FGF-21 has been described in U.S.Patent Application 20050176631, which is incorporated by referenceherein, to affect the overall metabolic state and may counter-actnegative side-effects that can occur during the body's stress responseto sepsis as well as systemic inflammatory response syndrome (SIRS)resulting from noninfectious pathologic causes. Thus, FGF-21 may be usedto reduce the mortality and morbidity that occurs in critically illpatients. Critically ill patients include those patients who arephysiologically unstable requiring continuous, coordinated physician,nursing, and respiratory care. This type of care necessitates payingparticular attention to detail in order to provide constant surveillanceand titration of therapy. Critically ill patients include those patientswho are at risk for physiological decompensation and thus requireconstant monitoring such that the intensive care team can provideimmediate intervention to prevent adverse occurrences. Critically illpatients have special needs for monitoring and life support which mustbe provided by a team that can provide continuous titrated care.

PEGylated FGF-21 polypeptides are described in WO 2005/091944, which isincorporated by reference herein. The FGF-21 polypeptide described in WO2005/091944 is a 181 amino acid polypeptide. The mature, wild-type, ornative human FGF-21 sequence indicated as SEQ ID NO: 1 of WO 2005/091944lacks a leader sequence. This human FGF-21 is highly identical to mouseFGF-21 (˜79% amino acid identity) and rat FGF-21 (˜80% amino acididentity). The human FGF-21 sequence presented as SEQ ID NO: 5 herein isthe same sequence as SEQ ID NO: 1 of WO 05/091944. This sequence has asingle nucleotide polymorphism (SNP) with leucine (L) at position 146.One of ordinary skill in the art could readily use alternative mammalianFGF-21 polypeptide sequences or analogs, muteins, or derivatives thathave sufficient homology to the human FGF-21 sequences for the usesdescribed herein.

The human FGF-21 sequence presented as SEQ ID NO: 1 herein has a singlenucleotide polymorphism (SNP) with proline (P) at position 146. AN-terminal His tag version of SEQ ID NO: 1 is shown as SEQ ID NO: 2herein.

WO 2005/091944 describes the covalent attachment of one or moremolecules of PEG to particular residues of an FGF-21 compound. Theresulting compound was a biologically active, PEGylated FGF-21 compoundwith an extended elimination half-life and reduced clearance whencompared to that of native FGF-21. The PEG molecules were covalentlyattached to cysteine or lysine residues. Substitutions were made atvarious positions with cysteine to allow attachment of at least one PEGmolecule. PEGylation at one or more lysine residues (56, 59, 69, and122) was described.

PEGylated FGF-21 compounds would be useful in treating subjects withdisorders, including, but not limited to, type 2 diabetes, obesity,insulin resistance, hyperinsulinemia, glucose intolerance,hyperglycemia, and metabolic syndrome. It would be particularlyadvantageous to have PEGylated FGF-21 compounds that could increaseefficacy by allowing for a longer circulating half-life and that wouldrequire fewer doses, increasing both the convenience to a subject inneed of such therapy and the likelihood of a subject's compliance withdosing requirements. Metabolic syndrome can be defined as a cluster ofat least three of the following signs: abdominal fat—in most men, a40-inch waist or greater; high blood sugar—at least 110 milligrams perdeciliter (mg/dL) after fasting; high triglycerides—at least 150 mg/dLin the bloodstream; low HDL—less than 40 mg/dL; and, blood pressure of130/85 of higher.

Covalent attachment of the hydrophilic polymer poly(ethylene glycol),abbreviated PEG, is a method of increasing water solubility,bioavailability, increasing serum half-life, increasing therapeutichalf-life, modulating immunogenicity, modulating biological activity, orextending the circulation time of many biologically active molecules,including proteins, peptides, and particularly hydrophobic molecules.PEG has been used extensively in pharmaceuticals, on artificialimplants, and in other applications where biocompatibility, lack oftoxicity, and lack of immunogenicity are of importance. In order tomaximize the desired properties of PEG, the total molecular weight andhydration state of the PEG polymer or polymers attached to thebiologically active molecule must be sufficiently high to impart theadvantageous characteristics typically associated with PEG polymerattachment, such as increased water solubility and circulating halflife, while not adversely impacting the bioactivity of the parentmolecule.

PEG derivatives are frequently linked to biologically active moleculesthrough reactive chemical functionalities, such as lysine, cysteine andhistidine residues, the N-terminus and carbohydrate moieties. There hasbeen research on the formulation of a therapeutic FGF-21 compound, butit has been problematic for many reasons, one of which is becauseproteins and other molecules often have a limited number of reactivesites available for polymer attachment. Often, the sites most suitablefor modification via polymer attachment play a significant role inreceptor binding, and are necessary for retention of the biologicalactivity of the molecule. As a result, indiscriminate attachment ofpolymer chains to such reactive sites on a biologically active moleculeoften leads to a significant reduction or even total loss of biologicalactivity of the polymer-modified molecule. R. Clark et al., (1996), J.Biol. Chem., 271:21969-21977. To form conjugates having sufficientpolymer molecular weight for imparting the desired advantages to atarget molecule, prior art approaches have typically involved randomattachment of numerous polymer arms to the molecule, thereby increasingthe risk of a reduction or even total loss in bioactivity of the parentmolecule.

Reactive sites that form the loci for attachment of PEG derivatives toproteins are dictated by the protein's structure. Proteins, includingenzymes, are composed of various sequences of alpha-amino acids, whichhave the general structure H₂N—CHR—COOH. The alpha amino moiety (H₂N—)of one amino acid joins to the carboxyl moiety (—COOH) of an adjacentamino acid to form amide linkages, which can be represented as—(NH—CHR—CO)_(n)—, where the subscript “n” can equal hundreds orthousands. The fragment represented by R can contain reactive sites forprotein biological activity and for attachment of PEG derivatives.

For example, in the case of the amino acid lysine, there exists an —NH₂moiety in the epsilon position as well as in the alpha position. Theepsilon —NH₂ is free for reaction under conditions of basic pH. Much ofthe art in the field of protein derivatization with PEG has beendirected to developing PEG derivatives for attachment to the epsilon—NH₂ moiety of lysine residues present in proteins. “Polyethylene Glycoland Derivatives for Advanced PEGylation”, Nektar Molecular EngineeringCatalog, 2003, pp. 1-17. These PEG derivatives all have the commonlimitation, however, that they cannot be installed selectively among theoften numerous lysine residues present on the surfaces of proteins. Thiscan be a significant limitation in instances where a lysine residue isimportant to protein activity, existing in an enzyme active site forexample, or in cases where a lysine residue plays a role in mediatingthe interaction of the protein with other biological molecules, as inthe case of receptor binding sites.

A second and equally important complication of existing methods forprotein PEGylation is that the PEG derivatives can undergo undesiredside reactions with residues other than those desired. Histidinecontains a reactive imino moiety, represented structurally as —N(H)—,but many chemically reactive species that react with epsilon —NH₂ canalso react with —N(H)—. Similarly, the side chain of the amino acidcysteine bears a free sulfhydryl group, represented structurally as —SH.In some instances, the PEG derivatives directed at the epsilon —NH₂group of lysine also react with cysteine, histidine or other residues.This can create complex, heterogeneous mixtures of PEG-derivatizedbioactive molecules and risks destroying the activity of the bioactivemolecule being targeted. It would be desirable to develop PEGderivatives that permit a chemical functional group to be introduced ata single site within the protein that would then enable the selectivecoupling of one or more PEG polymers to the bioactive molecule atspecific sites on the protein surface that are both well-defined andpredictable.

In addition to lysine residues, considerable effort in the art has beendirected toward the development of activated PEG reagents that targetother amino acid side chains, including cysteine, histidine and theN-terminus. See, e.g., U.S. Pat. No. 6,610,281 which is incorporated byreference herein, and “Polyethylene Glycol and Derivatives for AdvancedPEGylation”, Nektar Molecular Engineering Catalog, 2003, pp. 1-17. Acysteine residue can be introduced site-selectively into the structureof proteins using site-directed mutagenesis and other techniques knownin the art, and the resulting free sulfhydryl moiety can be reacted withPEG derivatives that bear thiol-reactive functional groups. Thisapproach is complicated, however, in that the introduction of a freesulfhydryl group can complicate the expression, folding and stability ofthe resulting protein. Thus, it would be desirable to have a means tointroduce a chemical functional group into FGF-21 that enables theselective coupling of one or more PEG polymers to the protein whilesimultaneously being compatible with (i.e., not engaging in undesiredside reactions with) sulfhydryls and other chemical functional groupstypically found in proteins.

As can be seen from a sampling of the art, many of these derivativesthat have been developed for attachment to the side chains of proteins,in particular, the —NH₂ moiety on the lysine amino acid side chain andthe —SH moiety on the cysteine side chain, have proven problematic intheir synthesis and use. Some form unstable linkages with the proteinthat are subject to hydrolysis and therefore decompose, degrade, or areotherwise unstable in aqueous environments, such as in the bloodstream.Some form more stable linkages, but are subject to hydrolysis before thelinkage is formed, which means that the reactive group on the PEGderivative may be inactivated before the protein can be attached. Someare somewhat toxic and are therefore less suitable for use in vivo. Someare too slow to react to be practically useful. Some result in a loss ofprotein activity by attaching to sites responsible for the protein'sactivity. Some are not specific in the sites to which they will attach,which can also result in a loss of desirable activity and in a lack ofreproducibility of results. In order to overcome the challengesassociated with modifying proteins with poly(ethylene glycol) moieties,PEG derivatives have been developed that are more stable (e.g., U.S.Pat. No. 6,602,498, which is incorporated by reference herein) or thatreact selectively with thiol moieties on molecules and surfaces (e.g.,U.S. Pat. No. 6,610,281, which is incorporated by reference herein).There is clearly a need in the art for PEG derivatives that arechemically inert in physiological environments until called upon toreact selectively to form stable chemical bonds.

Recently, an entirely new technology in the protein sciences has beenreported, which promises to overcome many of the limitations associatedwith site-specific modifications of proteins. Specifically, newcomponents have been added to the protein biosynthetic machinery of theprokaryote Escherichia coli (E. coli) (e.g., L. Wang, et al., (2001),Science 292:498-500) and the eukaryote Saccharomyces cerevisiae (S.cerevisiae) (e.g., J. Chin et al., Science 301:964-7 (2003)), which hasenabled the incorporation of non-genetically encoded amino acids toproteins in vivo. A number of new amino acids with novel chemical,physical or biological properties, including photoaffinity labels andphotoisomerizable amino acids, photocrosslinking amino acids (see, e.g.,Chin, J. W., et al. (2002) Proc. Natl. Acad. Sci. U.S.A 99:11020-11024;and, Chin, J. W., et al., (2002) J. Am. Chem. Soc. 124:9026-9027), ketoamino acids, heavy atom containing amino acids, and glycosylated aminoacids have been incorporated efficiently and with high fidelity intoproteins in E. coli and in yeast in response to the amber codon, TAG,using this methodology. See, e.g., J. W. Chin et al., (2002), Journal ofthe American Chemical Society 124:9026-9027; J. W. Chin, & P. G.Schultz, (2002), ChemBioChem 3(11):1135-1137; J. W. Chin, et al.,(2002), PNAS United States of America 99:11020-11024; and, L. Wang, & P.G. Schultz, (2002), Chem. Comm., 1:1-11. All references are incorporatedby reference in their entirety. These studies have demonstrated that itis possible to selectively and routinely introduce chemical functionalgroups, such as ketone groups, alkyne groups and azide moieties, thatare not found in proteins, that are chemically inert to all of thefunctional groups found in the 20 common, genetically-encoded aminoacids and that may be used to react efficiently and selectively to formstable covalent linkages.

The ability to incorporate non-genetically encoded amino acids intoproteins permits the introduction of chemical functional groups thatcould provide valuable alternatives to the naturally-occurringfunctional groups, such as the epsilon —NH₂ of lysine, the sulfhydryl—SH of cysteine, the imino group of histidine, etc. Certain chemicalfunctional groups are known to be inert to the functional groups foundin the 20 common, genetically-encoded amino acids but react cleanly andefficiently to form stable linkages. Azide and acetylene groups, forexample, are known in the art to undergo a Huisgen [3+2] cycloadditionreaction in aqueous conditions in the presence of a catalytic amount ofcopper. See, e.g., Tornoe, et al., (2002) J. Org. Chem. 67:3057-3064;and, Rostovtsev, et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599. Byintroducing an azide moiety into a protein structure, for example, oneis able to incorporate a functional group that is chemically inert toamines, sulfhydryls, carboxylic acids, hydroxyl groups found inproteins, but that also reacts smoothly and efficiently with anacetylene moiety to form a cycloaddition product. Importantly, in theabsence of the acetylene moiety, the azide remains chemically inert andunreactive in the presence of other protein side chains and underphysiological conditions.

The present invention addresses, among other things, problems associatedwith the activity and production of FGF-21 polypeptides, and alsoaddresses the production of an FGF-21 polypeptide with improvedbiological or pharmacological properties, such as improved therapeutichalf-life.

SUMMARY OF THE INVENTION

This invention provides FGF-21 polypeptides comprising one or morenon-naturally encoded amino acids.

In some embodiments, the FGF-21 polypeptide comprises one or morepost-translational modifications. In some embodiments, the FGF-21polypeptide is linked to a linker, polymer, or biologically activemolecule. In some embodiments, the FGF-21 polypeptide is linked to abifunctional polymer, bifunctional linker, or at least one additionalFGF-21 polypeptide.

In some embodiments, the non-naturally encoded amino acid is linked to awater soluble polymer. In some embodiments, the water soluble polymercomprises a poly(ethylene glycol) moiety. In some embodiments, thenon-naturally encoded amino acid is linked to the water soluble polymerwith a linker or is bonded to the water soluble polymer. In someembodiments, the poly(ethylene glycol) molecule is a bifunctionalpolymer. In some embodiments, the bifunctional polymer is linked to asecond polypeptide. In some embodiments, the second polypeptide is aFGF-21 polypeptide.

In some embodiments, the FGF-21 polypeptide comprises at least two aminoacids linked to a water soluble polymer comprising a poly(ethyleneglycol) moiety. In some embodiments, at least one amino acid is anon-naturally encoded amino acid.

In some embodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in FGF-21: beforeposition 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 (i.e., at thecarboxyl terminus of the protein) (SEQ ID NO: 1 or the correspondingamino acids in SEQ ID NOs: 2-7). In some embodiments, one or morenon-naturally encoded amino acids are incorporated in one or morepositions from before position 1 (i.e. at the N-terminus) through the Cterminus in SEQ ID NOs: 34-36. In some embodiments, one or morenon-naturally encoded amino acids are incorporated in one or more of thefollowing positions in FGF-21: 10, 52, 117, 126, 131, 162, 87, 77, 83,72, 69, 79, 91, 96, 108, and 110 (SEQ ID NO: 1 or the correspondingamino acids of SEQ ID NOs: 2-7). In some embodiments, one or morenon-naturally encoded amino acids are incorporated in one or more of thefollowing positions in FGF-21: 10, 52, 77, 117, 126, 131, 162 (SEQ IDNO: 1 or the corresponding amino acids of SEQ ID NOs: 2-7). In someembodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in FGF-21: 87,77, 83, 72 (SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs:2-7). In some embodiments, one or more non-naturally encoded amino acidsare incorporated in one or more of the following positions in FGF-21:69, 79, 91, 96, 108, and 110 (SEQ ID NO: 1 or the corresponding aminoacids of SEQ ID NOs: 2-7). In some embodiments, one or more non-naturalamino acids are incorporated in the leader or signal sequence of SEQ IDNOs: 3, 4, 6, 7, or other FGF-21 sequence. In some embodiments, leadersequences may be chosen from SEQ ID NOs: 39, 40, 41, 42, 43, or 44. Insome embodiments, FGF-21 secretion constructs are cloned into pVK7ara(Nde/Eco) with a leader sequences chosen from SEQ ID NOs: 39, 40, 41,42, 43, or 44.

In some embodiments, the non-naturally occurring amino acid at one ormore of these positions is linked to a water soluble polymer, includingbut not limited to, positions: before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182 (i.e., at the carboxyl terminus of theprotein) (SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs:2-7). In some embodiments, the non-naturally occurring amino acid at oneor more positions from before position 1 (i.e. at the N-terminus)through the C terminus in SEQ ID NOs: 34-36 is linked to a water solublepolymer. In some embodiments, the non-naturally occurring amino acid atone or more of these positions is linked to a water soluble polymer,including but not limited to, positions: 10, 52, 117, 126, 131, 162, 87,77, 83, 72, 69, 79, 91, 96, 108, and 110 (SEQ ID NO: 1 or thecorresponding amino acids of SEQ ID NOs: 2-7). In some embodiments, thenon-naturally occurring amino acid at one or more of these positions islinked to a water soluble polymer, including but not limited to,positions: 10, 52, 77, 117, 126, 131, 162 (SEQ ID NO: 1 or thecorresponding amino acids of SEQ ID NOs: 2-7). In some embodiments, thenon-naturally occurring amino acid at one or more of these positions islinked to a water soluble polymer: 87, 77, 83, 72 (SEQ ID NO: 1 or thecorresponding amino acids of SEQ ID NOs: 2-7). In some embodiments, thenon-naturally occurring amino acid at one or more of these positions islinked to a water soluble polymer: 69, 79, 91, 96, 108, and 110 (SEQ IDNO: 1 or the corresponding amino acids of SEQ ID NOs: 2-7). In someembodiments, the one or more non-naturally occurring amino acids in theleader or signal sequence of SEQ ID NOs: 3, 4, 6, 7, 39, 40, 41, 42, 43,44, or other FGF-21 sequence is linked to a water soluble polymer. Insome embodiments, the one or more non-naturally occurring amino acids inthe leader or signal sequence of SEQ ID NOs: 3, 4, 6, 7, or other FGF-21sequence is linked to a water soluble polymer.

In some embodiments, the FGF-21 polypeptide comprises a substitution,addition or deletion that modulates affinity of the FGF-21 polypeptidefor a FGF-21 polypeptide receptor or binding partner, including but notlimited to, a protein, polypeptide, small molecule, or nucleic acid. Insome embodiments, the FGF-21 polypeptide comprises a substitution,addition, or deletion that increases the stability of the FGF-21polypeptide when compared with the stability of the corresponding FGF-21without the substitution, addition, or deletion. In some embodiments,the FGF-21 polypeptide comprises a substitution, addition, or deletionthat modulates the immunogenicity of the FGF-21 polypeptide whencompared with the immunogenicity of the corresponding FGF-21 without thesubstitution, addition, or deletion. In some embodiments, the FGF-21polypeptide comprises a substitution, addition, or deletion thatmodulates serum half-life or circulation time of the FGF-21 polypeptidewhen compared with the serum half-life or circulation time of thecorresponding FGF-21 without the substitution, addition, or deletion.

In some embodiments, the FGF-21 polypeptide comprises a substitution,addition, or deletion that increases the aqueous solubility of theFGF-21 polypeptide when compared to aqueous solubility of thecorresponding FGF-21 without the substitution, addition, or deletion. Insome embodiments, the FGF-21 polypeptide comprises a substitution,addition, or deletion that increases the solubility of the FGF-21polypeptide produced in a host cell when compared to the solubility ofthe corresponding FGF-21 without the substitution, addition, ordeletion. In some embodiments, the FGF-21 polypeptide comprises asubstitution, addition, or deletion that increases the expression of theFGF-21 polypeptide in a host cell or increases synthesis in vitro whencompared to the expression or synthesis of the corresponding FGF-21without the substitution, addition, or deletion. The FGF-21 polypeptidecomprising this substitution retains agonist activity and retains orimproves expression levels in a host cell. In some embodiments, theFGF-21 polypeptide comprises a substitution, addition, or deletion thatincreases protease resistance of the FGF-21 polypeptide when compared tothe protease resistance of the corresponding FGF-21 without thesubstitution, addition, or deletion. U.S. Pat. No. 6,716,626 indicatedthat potential sites that may be substituted to alter protease cleavageinclude, but are not limited to, a monobasic site within 2 residues of aproline. In some embodiments, the FGF-21 polypeptide comprises asubstitution, addition, or deletion that modulates signal transductionactivity of the FGF-21 receptor when compared with the activity of thereceptor upon interaction with the corresponding FGF-21 polypeptidewithout the substitution, addition, or deletion. In some embodiments,the FGF-21 polypeptide comprises a substitution, addition, or deletionthat modulates its binding to another molecule such as a receptor whencompared to the binding of the corresponding FGF-21 polypeptide withoutthe substitution, addition, or deletion.

In some embodiments, the FGF-21 polypeptide comprises a substitution,addition, or deletion that increases compatibility of the FGF-21polypeptide with pharmaceutical preservatives (e.g., m-cresol, phenol,benzyl alcohol) when compared to compatibility of the correspondingFGF-21 without the substitution, addition, or deletion. This increasedcompatibility would enable the preparation of a preserved pharmaceuticalformulation that maintains the physiochemical properties and biologicalactivity of the protein during storage. WO 2005/091944, which isincorporated by reference in its entirety, discusses the followingexamples of FGF-21 muteins with enhanced pharmaceutical stability: thesubstitution with a charged and/or polar but uncharged amino acid forone of the following: glycine 42, glutamine 54, arginine 77, alanine 81,leucine 86, phenylalanine 88, lysine 122, histidine 125, arginine 126,proline 130, arginine 131, leucine 139, alanine 145, leucine 146,isoleucine 152, alanine 154, glutamine 156, glycine 161, serine 163,glycine 170, or serine 172 of SEQ ID NO: 1 of WO 05/091944. A FGF-21polypeptide of the present invention may include one or more of thesesubstitutions at the corresponding position in the polypeptide and/ormay include one or more other substitutions, additions, or deletions. Insome embodiments, one or more non-natural amino acids are substituted atone or more of the following positions: glycine 42, glutamine 54,arginine 77, alanine 81, leucine 86, phenylalanine 88, lysine 122,histidine 125, arginine 126, proline 130, arginine 131, leucine 139,alanine 145, proline/leucine 146, isoleucine 152, alanine 154, glutamine156, glycine 161, serine 163, glycine 170, serine 172 (SEQ ID NO: 1 orthe corresponding amino acids in SEQ ID NOs: 2-7). In some embodiments,one or more non-natural amino acids are substituted at one or more ofthe following positions: glutamate 91, arginine 131, glutamine 108,arginine 77, arginine 72, histidine 87, leucine 86, arginine 126,glutamate 110, tyrosine 83, proline 146, arginine 135, arginine 96,arginine 36, (SEQ ID NO: 1 or the corresponding amino acids in SEQ IDNOs: 2-7).

WO 05/091944 describes additional muteins of FGF-21 with enhancedpharmaceutical stability. Such muteins include the substitution of acysteine for two or more of the following in FGF-21 (see SEQ ID NO: 1 ofWO 05/091944): arginine 19, tyrosine 20, leucine 21, tyrosine 22,threonine 23, aspartate 24, aspartate 25, alanine 26, glutamine 27,glutamine 28, alanine 31, leucine 33, isoleucine 35, leucine 37, valine41, glycine 42, glycine 43, glutamate 50, glutamine 54, leucine 58,valine 62, leucine 66, glycine 67, lysine 69, arginine 72, phenylalanine73, glutamine 76, arginine 77, aspartate 79, glycine 80, alanine 81,leucine 82, glycine 84, serine 85, proline 90, alanine 92, serine 94,phenylalanine 95, leucine 100, aspartate 102, tyrosine 104, tyrosine107, serine 109, glutamate 110, proline 115, histidine 117, leucine 118,proline 119, asparagine 121, lysine 122, serine 123, proline 124,histidine 125, arginine 126, aspartate 127, alanine 129, proline 130,glycine 132, alanine 134, arginine 135, leucine 137, proline 138, orleucine 139. FGF-21 polypeptides of the present invention may includeone or more of these substitutions at the corresponding position in thepolypeptide and/or may include one or more other substitutions,additions, or deletions. In some embodiments, one or more non-naturalamino acids are substituted at one or more of the following positions:arginine 19, tyrosine 20, leucine 21, tyrosine 22, threonine 23,aspartate 24, aspartate 25, alanine 26, glutamine 27, glutamine 28,alanine 31, leucine 33, isoleucine 35, leucine 37, valine 41, glycine42, glycine 43, glutamate 50, glutamine 54, leucine 58, valine 62,leucine 66, glycine 67, lysine 69, arginine 72, phenylalanine 73,glutamine 76, arginine 77, aspartate 79, glycine 80, alanine 81, leucine82, glycine 84, serine 85, proline 90, alanine 92, serine 94,phenylalanine 95, leucine 100, aspartate 102, tyrosine 104, tyrosine107, serine 109, glutamate 110, proline 115, histidine 117, leucine 118,proline 119, asparagine 121, lysine 122, serine 123, proline 124,histidine 125, arginine 126, aspartate 127, alanine 129, proline 130,glycine 132, alanine 134, arginine 135, leucine 137, proline 138, orleucine 139 (SEQ ID NO: 1 or the corresponding amino acids in SEQ IDNOs: 2-7).

WO 05/091944 further describes specific muteins of FGF-21 withengineered disulfide bonds (amino acids substituted with cysteine), inaddition to the naturally occurring one at Cys75-Cys93, are as follows:Gln76Cys-Ser109Cys, Cys75-Ser85Cys, Cys75-Ala92Cys, Phe73Cys-Cys93,Ser123Cys-His125Cys, Asp102Cys-Tyr104Cys, Asp127Cys-Glyl32Cys,Ser94Cys-Glu110Cys, Pro115Cys-His117Cys, Asn121Cys-Asp127Cys,Leu100Cys-Asp102Cys, Phe95Cys-Tyr107Cys, Arg19CysPro138Cys,Tyr20Cys-Leu139Cys, Tyr22Cys-Leu137Cys, Arg77Cys-Asp79Cys,Pro90Cys-Ala92Cys, Glu50Cys-Lys69Cys, Thr23Cys-Asp25Cys,Ala31Cys-Gly43Cys, Gln28Cys-Gly43Cys, Thr23Cys-G1n28Cys,Va141Cys-Leu82Cys, Leu58Cys-Va162Cys, Gln54Cys-Leu66Cys,Ile35Cys-Gly67Cys, Gly67Cys-Arg72Cys, Ile35Cys-Gly84Cys,Arg72Cys-Gly84Cys, or Arg77Cys-Ala81Cys, where the numbering is based onSEQ ID NO: 1 of WO 05/091944. Additional muteins with engineereddisulfide bonds are Tyr22Cys-Leu139Cys; Asp24Cys-Arg135Cys;Leu118Cys-Glyl32Cys; His117Cys-Pro130Cys; His117Cys-Ala129Cys;Leu82Cys-Pro119Cys; Gly80Cys-Ala129Cys; Gly43Cys-Pro124Cys;Gly42Cys-Arg126Cys; Gly42Cys-Pro124Cys; Gln28Cys-Pro124Cys;Gln27Cys-Ser123Cys; Ala26Cys-Lys122Cys; or Asp25Cys-Lys122Cys, where thenumbering is based on SEQ ID NO: 1 of WO 05/091944. Additional mutienswith engineered disulfide bonds are Leu118Cys-Ala134Cys;Leu21Cys-Leu33Cys; Ala26Cys-Lys122Cys;Leu21Cys-Leu33Cys/Leu118Cys-Ala134Cys, where the numbering is based onSEQ ID NO: 1 of WO 05/091944. FGF-21 polypeptides of the presentinvention may include one or more of these substitutions at thecorresponding position(s) in the polypeptide and/or may include one ormore other substitutions, additions, or deletions. FGF-21 polypeptidesof the present invention may include one or more of these substitutionsat the corresponding position(s) in the polypeptide (SEQ ID NO: 1 or thecorresponding amino acids in SEQ ID NOs: 2-7). In some embodiments,FGF-21 polypeptides of the present invention may include one or more ofthese substitutions at the corresponding positions from before position1 (i.e. at the N-terminus) through the C terminus in SEQ ID NOs: 34-36.

WO 05/091944 describes additional muteins of FGF-21 that were PEGylated.These muteins had one of the following substitutions: D25C, D38C, L58C,K59C, P60C, K69C, D79C, H₈₇C, E91C, E101C, D102C, L114C, L116C, K122C,R126C, P130C, P133C, P140C. FGF-21 polypeptides of the present inventionmay include one or more of these substitutions at the correspondingposition in the polypeptide and/or may include one or more othersubstitutions, additions, or deletions. In some embodiments, one or morenon-natural amino acids are substituted at one or more of the followingpositions: 25, 38, 58, 59, 60, 69, 79, 87, 91, 101, 102, 114, 116, 122,126, 130, 133, 140 (SEQ ID NO: 1 or the corresponding amino acids in SEQID NOs: 2-7). In some embodiments, FGF-21 polypeptides of the presentinvention may include one or more of these substitutions at thecorresponding positions from before position 1 (i.e. at the N-terminus)through the C terminus in SEQ ID NOs: 34-36.

WO 05/091944 describes cysteine substitutions at the followingpositions: 19, 21, 26, 28, 29, 30, 36, 39, 42, 50, 56, 61, 64, 65, 68,70, 71, 77, 81, 85, 86, 90, 92, 94, 98, 107, 108, 112, 113, 123, and124. WO 05/091944 indicates cysteine substitutions at the followingpositions: 24, 27, 37, 40, 44, 46, 49, 57, 88, 89, 106, 110, 111, 115,120, and 139. WO 05/091944 also describes cysteine substitutions at thefollowing positions: 18, 45, 47, 48, 78, 83, 99, 103, 125, 128, 131,132, and 138. WO 05/091944 also describes cysteine substitutions at thefollowing positions: 25, 38, 58, 59, 60, 69, 79, 87, 91, 101, 102, 114,116, 122, 126, 130, 133, and 140.

In some embodiments, one or more engineered bonds are created with oneor more non-natural amino acids. The intramolecular bond may be createdin many ways, including but not limited to, a reaction between two aminoacids in the protein under suitable conditions (one or both amino acidsmay be a non-natural amino acid); a reaction with two amino acids, eachof which may be naturally encoded or non-naturally encoded, with alinker, polymer, or other molecule under suitable conditions; etc.

In some embodiments, one or more amino acid substitutions in the FGF-21polypeptide may be with one or more naturally occurring or non-naturallyoccurring amino acids. In some embodiments the amino acid substitutionsin the FGF-21 polypeptide may be with naturally occurring ornon-naturally occurring amino acids, provided that at least onesubstitution is with a non-naturally encoded amino acid. In someembodiments, one or more amino acid substitutions in the FGF-21polypeptide may be with one or more naturally occurring amino acids, andadditionally at least one substitution is with a non-naturally encodedamino acid.

In some embodiments, the non-naturally encoded amino acid comprises acarbonyl group, an acetyl group, an aminooxy group, a hydrazine group, ahydrazide group, a semicarbazide group, an azide group, or an alkynegroup.

In some embodiments, the non-naturally encoded amino acid comprises acarbonyl group. In some embodiments, the non-naturally encoded aminoacid has the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; R₂ is H, an alkyl, aryl, substituted alkyl, andsubstituted aryl; and R₃ is H, an amino acid, a polypeptide, or an aminoterminus modification group, and R₄ is H, an amino acid, a polypeptide,or a carboxy terminus modification group.

In some embodiments, the non-naturally encoded amino acid comprises anaminooxy group. In some embodiments, the non-naturally encoded aminoacid comprises a hydrazide group. In some embodiments, the non-naturallyencoded amino acid comprises a hydrazine group. In some embodiments, thenon-naturally encoded amino acid residue comprises a semicarbazidegroup.

In some embodiments, the non-naturally encoded amino acid residuecomprises an azide group. In some embodiments, the non-naturally encodedamino acid has the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, substitutedaryl or not present; X is O, N, S or not present; m is 0-10; R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, the non-naturally encoded amino acid comprises analkyne group. In some embodiments, the non-naturally encoded amino acidhas the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; X is O, N, S or not present; m is 0-10, R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, the polypeptide is a FGF-21 polypeptide agonist,partial agonist, antagonist, partial antagonist, or inverse agonist. Insome embodiments, the FGF-21 polypeptide agonist, partial agonist,antagonist, partial antagonist, or inverse agonist comprises anon-naturally encoded amino acid linked to a water soluble polymer. Insome embodiments, the water soluble polymer comprises a poly(ethyleneglycol) moiety. In some embodiments, the FGF-21 polypeptide agonist,partial agonist, antagonist, partial antagonist, or inverse agonistcomprises a non-naturally encoded amino acid and one or morepost-translational modification, linker, polymer, or biologically activemolecule.

The present invention also provides isolated nucleic acids comprising apolynucleotide that hybridizes under stringent conditions to SEQ ID NO:8-14. The present invention also provides isolated nucleic acidscomprising a polynucleotide that hybridizes under stringent conditionsto SEQ ID NO: 8-14 wherein the polynucleotide comprises at least oneselector codon. The present invention also provides isolated nucleicacids comprising a polynucleotide that encodes the polypeptides shown asSEQ ID NOs.: 1-7. The present invention also provides isolated nucleicacids comprising a polynucleotide that encodes the polypeptides shown asSEQ ID NOs.: 1-7 with one or more non-naturally encoded amino acids. Itis readily apparent to those of ordinary skill in the art that a numberof different polynucleotides can encode any polypeptide of the presentinvention.

In some embodiments, the selector codon is selected from the groupconsisting of an amber codon, ochre codon, opal codon, a unique codon, arare codon, a five-base codon, and a four-base codon.

The present invention also provides methods of making a FGF-21polypeptide linked to a water soluble polymer. In some embodiments, themethod comprises contacting an isolated FGF-21 polypeptide comprising anon-naturally encoded amino acid with a water soluble polymer comprisinga moiety that reacts with the non-naturally encoded amino acid. In someembodiments, the non-naturally encoded amino acid incorporated into theFGF-21 polypeptide is reactive toward a water soluble polymer that isotherwise unreactive toward any of the 20 common amino acids. In someembodiments, the non-naturally encoded amino acid incorporated into theFGF-21 polypeptide is reactive toward a linker, polymer, or biologicallyactive molecule that is otherwise unreactive toward any of the 20 commonamino acids.

In some embodiments, the FGF-21 polypeptide linked to the water solublepolymer is made by reacting a FGF-21 polypeptide comprising acarbonyl-containing amino acid with a poly(ethylene glycol) moleculecomprising an aminooxy, hydrazine, hydrazide or semicarbazide group. Insome embodiments, the aminooxy, hydrazine, hydrazide or semicarbazidegroup is linked to the poly(ethylene glycol) molecule through an amidelinkage.

In some embodiments, the FGF-21 polypeptide linked to the water solublepolymer is made by reacting a poly(ethylene glycol) molecule comprisinga carbonyl group with a polypeptide comprising a non-naturally encodedamino acid that comprises an aminooxy, hydrazine, hydrazide orsemicarbazide group.

In some embodiments, the FGF-21 polypeptide linked to the water solublepolymer is made by reacting a FGF-21 polypeptide comprising analkyne-containing amino acid with a poly(ethylene glycol) moleculecomprising an azide moiety. In some embodiments, the azide or alkynegroup is linked to the poly(ethylene glycol) molecule through an amidelinkage.

In some embodiments, the FGF-21 polypeptide linked to the water solublepolymer is made by reacting a FGF-21 polypeptide comprising anazide-containing amino acid with a poly(ethylene glycol) moleculecomprising an alkyne moiety. In some embodiments, the azide or alkynegroup is linked to the poly(ethylene glycol) molecule through an amidelinkage.

In some embodiments, the poly(ethylene glycol) molecule has a molecularweight of between about 0.1 kDa and about 100 kDa. In some embodiments,the poly(ethylene glycol) molecule has a molecular weight of between 0.1kDa and 50 kDa.

In some embodiments, the poly(ethylene glycol) molecule is a branchedpolymer. In some embodiments, each branch of the poly(ethylene glycol)branched polymer has a molecular weight of between 1 kDa and 100 kDa, orbetween 1 kDa and 50 kDa.

In some embodiments, the water soluble polymer linked to the FGF-21polypeptide comprises a polyalkylene glycol moiety. In some embodiments,the non-naturally encoded amino acid residue incorporated into theFGF-21 polypeptide comprises a carbonyl group, an aminooxy group, ahydrazide group, a hydrazine, a semicarbazide group, an azide group, oran alkyne group. In some embodiments, the non-naturally encoded aminoacid residue incorporated into the FGF-21 polypeptide comprises acarbonyl moiety and the water soluble polymer comprises an aminooxy,hydrazide, hydrazine, or semicarbazide moiety. In some embodiments, thenon-naturally encoded amino acid residue incorporated into the FGF-21polypeptide comprises an alkyne moiety and the water soluble polymercomprises an azide moiety. In some embodiments, the non-naturallyencoded amino acid residue incorporated into the FGF-21 polypeptidecomprises an azide moiety and the water soluble polymer comprises analkyne moiety.

The present invention also provides compositions comprising a FGF-21polypeptide comprising a non-naturally encoded amino acid and apharmaceutically acceptable carrier. In some embodiments, thenon-naturally encoded amino acid is linked to a water soluble polymer.

The present invention also provides cells comprising a polynucleotideencoding the FGF-21 polypeptide comprising a selector codon. In someembodiments, the cells comprise an orthogonal RNA synthetase and/or anorthogonal tRNA for substituting a non-naturally encoded amino acid intothe FGF-21 polypeptide.

The present invention also provides methods of making a FGF-21polypeptide comprising a non-naturally encoded amino acid. In someembodiments, the methods comprise culturing cells comprising apolynucleotide or polynucleotides encoding a FGF-21 polypeptide, anorthogonal RNA synthetase and/or an orthogonal tRNA under conditions topermit expression of the FGF-21 polypeptide; and purifying the FGF-21polypeptide from the cells and/or culture medium.

The present invention also provides methods of increasing therapeutichalf-life, serum half-life or circulation time of FGF-21 polypeptides.The present invention also provides methods of modulating immunogenicityof FGF-21 polypeptides. In some embodiments, the methods comprisesubstituting a non-naturally encoded amino acid for any one or moreamino acids in naturally occurring FGF-21 polypeptides and/or linkingthe FGF-21 polypeptide to a linker, a polymer, a water soluble polymer,or a biologically active molecule.

The present invention also provides methods of treating a patient inneed of such treatment with an effective amount of a FGF-21 molecule ofthe present invention. In some embodiments, the methods compriseadministering to the patient a therapeutically-effective amount of apharmaceutical composition comprising a FGF-21 polypeptide comprising anon-naturally-encoded amino acid and a pharmaceutically acceptablecarrier. In some embodiments, the non-naturally encoded amino acid islinked to a water soluble polymer.

The present invention also provides FGF-21 polypeptides comprising asequence shown in SEQ ID NO: 1-7 or any other FGF-21 polypeptidesequence, except that at least one amino acid is substituted by anon-naturally encoded amino acid. The present invention also providesFGF-21 polypeptides comprising a sequence shown as SEQ ID NO: 1, 2, 4,and 5. In some embodiments, the non-naturally encoded amino acid islinked to a water soluble polymer. In some embodiments, the watersoluble polymer comprises a poly(ethylene glycol) moiety. In someembodiments, the non-naturally encoded amino acid comprises a carbonylgroup, an aminooxy group, a hydrazide group, a hydrazine group, asemicarbazide group, an azide group, or an alkyne group.

The present invention also provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a FGF-21polypeptide comprising the sequence shown in SEQ ID NO: 1-7 or any otherFGF-21 polypeptide sequence, wherein at least one amino acid issubstituted by a non-naturally encoded amino acid. The present inventionalso provides pharmaceutical compositions comprising a pharmaceuticallyacceptable carrier and a FGF polypeptide comprising the sequence shownin SEQ ID NO: 1-7.In some embodiments, the non-naturally encoded aminoacid comprises a saccharide moiety. In some embodiments, the watersoluble polymer is linked to the polypeptide via a saccharide moiety. Insome embodiments, a linker, polymer, or biologically active molecule islinked to the FGF-21 polypeptide via a saccharide moiety.

The present invention also provides a FGF-21 polypeptide comprising awater soluble polymer linked by a covalent bond to the FGF-21polypeptide at a single amino acid. In some embodiments, the watersoluble polymer comprises a poly(ethylene glycol) moiety. In someembodiments, the amino acid covalently linked to the water solublepolymer is a non-naturally encoded amino acid present in thepolypeptide.

The present invention provides a FGF-21 polypeptide comprising at leastone linker, polymer, or biologically active molecule, wherein saidlinker, polymer, or biologically active molecule is attached to thepolypeptide through a functional group of a non-naturally encoded aminoacid ribosomally incorporated into the polypeptide. In some embodiments,the polypeptide is monoPEGylated. The present invention also provides aFGF-21 polypeptide comprising a linker, polymer, or biologically activemolecule that is attached to one or more non-naturally encoded aminoacid wherein said non-naturally encoded amino acid is ribosomallyincorporated into the polypeptide at pre-selected sites.

Included within the scope of this invention is the FGF-21 leader orsignal sequence joined to an FGF-21 coding region, as well as aheterologous signal sequence joined to an FGF-21 coding region. Theheterologous leader or signal sequence selected should be one that isrecognized and processed, e.g. by host cell secretion system to secreteand possibly cleaved by a signal peptidase, by the host cell. Leadersequences of the present invention may be chosen from the following: thethree leucine leader from SEQ ID NO: 3 and SEQ ID NO: 6 (amino acidpositions 1-28), the two leucine leader from SEQ ID NO: 4 and SEQ ID NO:7 (amino acid positions 1-27), the His tag from SEQ ID NO: 2 (amino acidpositions 1-10), SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO:42, SEQ ID NO: 43, SEQ ID NO: 44. A method of treating a condition ordisorder with the FGF-21 of the present invention is meant to implytreating with FGF-21 with or without a signal or leader peptide.

The present invention also provides methods of inducing an increase inglucose uptake in adipocyte cells, said method comprising administeringFGF-21 to said cells in an amount effective to induce an increase inglucose uptake. Said increase in glucose uptake may cause an increase inenergy expenditure by faster and more efficient glucose utilization.

In another embodiment, conjugation of the FGF-21 polypeptide comprisingone or more non-naturally occurring amino acids to another molecule,including but not limited to PEG, provides substantially purified FGF-21due to the unique chemical reaction utilized for conjugation to thenon-natural amino acid. Conjugation of FGF-21 comprising one or morenon-naturally encoded amino acids to another molecule, such as PEG, maybe performed with other purification techniques performed prior to orfollowing the conjugation step to provide substantially pure FGF-21.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1—Amber mutations in FGF-21 and corresponding sites in FGF-19 areshown.

FIG. 2—The structure of human FGF-19 is shown.

FIG. 3—Amber mutations in FGF-21 and corresponding sites in FGF-2 areshown.

FIG. 4—The structure of human FGF-19 is shown.

FIG. 5—Expression of N-terminal His tagged FGF-21 and suppression at 7amber sites are shown.

FIG. 6—BPER supernatant samples from the expression of N-terminal Histagged FGF-21 and suppression at 7 amber sites are shown.

FIG. 7a —SigmaPlot calculating the EC50 values for serial dilutions ofFGF21 variants 30K PEG-391, 30K PEG-477, 30K PEG-R131, 30K PEG-Q108,HIS-FGF21 (His-tagged wild type).

FIG. 7b —A table showing the average fold loss of activity for each ofthe pegylated FGF21 variants listed.

FIG. 8—An SDS-PAGE analysis of non-His-tagged FGF-21 expressed in E.coli.

FIGS. 9A-C—FIG. 9A: SDS-PAGE analysis of FGF-21-Y83pAF elutionfractions.

FIG. 9B Chromatogram of Q HP elution of untagged FGF-21-Y83pAF. FIG. 9CSDS-PAGE analysis of FGF-21-Y832pAFQ HP elution pool.

FIG. 10—Data from Example 28, Pharmacokinetic properties of FGF-21compounds in rats.

FIG. 11—Data from Example 28, Pharmacokinetic properties of FGF-21compounds in rats.

FIG. 12—Data from Example 28, Pharmacokinetic properties of FGF-21compounds in rats.

FIG. 13—Data from Example 28, Pharmacokinetic properties of FGF-21compounds in rats.

FIG. 14—Data from Example 28, Pharmacokinetic properties of FGF-21compounds in rats.

FIG. 15—Data from Example 28, Pharmacokinetic properties of FGF-21compounds in rats.

FIG. 16—Data from Example 28, Pharmacokinetic properties of FGF-21compounds in rats.

FIG. 17—Data from Example 28, Pharmacokinetic properties of FGF-21compounds in rats.

FIG. 18—Data from Example 28, Pharmacokinetic properties of FGF-21compounds in rats.

FIG. 19—Data from Example 28, Pharmacokinetic properties of FGF-21compounds in rats.

FIG. 20—Data from Example 28, Pharmacokinetic properties of FGF-21compounds in rats.

FIG. 21—Data from Example 28, Pharmacokinetic properties of FGF-21compounds in rats.

FIG. 22—Data from Example 28, Pharmacokinetic properties of FGF-21compounds in rats.

FIG. 23—Data from Example 28, Pharmacokinetic properties of FGF-21compounds in rats.

FIG. 24—pVK10-FGF21 vector map.

FIG. 25—pVK10-FGF21 vector sequence.

FIG. 26a -Serum concentration-time profiles of three doses of N-6His WTFGF21 in rats. Rats were given a single administration of test articlesubcutaneously. N=4 animals per group. Symbols indicate means ofmeasured serum concentrations, error bars indicate standard error.

FIG. 26b -Serum concentration-time profiles of N-6His WT FGF21 dosedeither subcutaneously or intravenously at 0.25 mg/kg. Rats were given asingle administration of test article subcutaneously. N=4 animals pergroup. Symbols indicate means of measured serum concentrations, errorbars indicate standard error. Total bioavailability is ˜87%

FIG. 27a -Dose relationship to serum concentration of test article atCmax. Cmax values are reported as observed not theoretical. N=4 animalsper treatment group. The linear regression value is 0.59 with a slope of348.5±91.22.

FIG. 27b -Dose relationship to terminal half-life of test article. N=4animals per treatment group. The linear regression value could not becalculated due to an apparent saturation of clearance above 0.25 mg/kg.

FIG. 27c -Dose relationship to serum concentration AUC. AUC values arereported as observed calculated to infinity. N=4 animals per treatmentgroup. The linear regression value is 0.75 with a slope of 1079±194.1

FIG. 28a -Serum concentration-time profiles of three doses of PP WTFGF21 in rats. Rats were given a single administration of test articlesubcutaneously. N=4 animals per group. Symbols indicate means ofmeasured serum concentrations, error bars indicate standard error.

FIG. 28b -Serum concentration-time profiles of PP WT FGF21 dosed eithersubcutaneously or intravenously at 0.25 mg/kg. Rats were given a singleadministration of test article subcutaneously. N=4 animals per group.Symbols indicate means of measured serum concentrations, error barsindicate standard error. The total bioavailability is ˜65%

FIG. 29a -Dose relationship to serum concentration of test article atCmax. Cmax values are reported as observed not theoretical. N=4 animalsper treatment group. The linear regression value is 0.92 with a slope of454.2±42.42.

FIG. 29b -Dose relationship to terminal half-life of test article. N=4animals per treatment group. The linear regression value could not becalculated due to an apparent saturation of clearance above 0.125 mg/kg.

FIG. 29c -Dose relationship to serum concentration AUC. AUC values arereported as observed calculated to infinity. N=4 animals per treatmentgroup. The linear regression value is 0.93 with a slope of 1585±137.1

FIG. 30a -Comparison of calculated terminal half-life for PP versusN6-His WT FGF21 compounds dosed at 0.5 mg/kg subcutaneously in rats. Thecalculated p value using a two-tailed t-test is 0.7715. N=3-4 animalsper group

FIG. 30b -Comparison of Cmax values for PP versus N6-His WT FGF21compounds dosed at 0.5 mg/kg subcutaneously in rats. The calculated pvalue using a two-tailed t-test is 0.7652. N=3-4 animals per group

FIG. 30c -Comparison of AUCinf for PP versus N6-His WT FGF21 compoundsdosed at 0.5 mg/kg subcutaneously in rats. The calculated p value usinga two-tailed t-test is 0.4372

FIG. 31a -PK profiles of ten PEGylated N6-His tagged FGF21 isomers.

FIG. 31b -Absorption profiles for PEGylated FGF21 isomers after 0.25mg/kg subcutaneous injection.

FIG. 31c -Elimination profiles for PEGylated FGF21 isomers after 0.25mg/kg subcutaneous injection.

FIG. 32—Pharmacokinetic comparison of 20 and 30 kDa PEGylation.

FIG. 33—Plasma concentration time curves for rats dosed eitherintravenously or subcutaneously with 0.25 mg/kg of20KPEG-pAF91(N6-His)FGF21. A single dose was administered to eachanimal. N=4 animals per group. Symbols indicate means of measured plasmaconcentrations, bars indicate standard deviation. Total bioavailabilityis ˜30%.

FIG. 34—Two gels showing the secretion of FGF21 in e.coli and showing ofthe leaders used that OmpA, MalE, and RH worked very well, asdemonstrated by the periplasmic release soluble fraction in the secondgel.

DEFINITIONS

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, constructs, and reagentsdescribed herein and as such may vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention, which will be limited only by the appended claims.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. Thus, for example, reference to a “FGF-21” or “FGF-21polypeptide” is a reference to one or more such proteins and includesequivalents thereof known to those of ordinary skill in the art, and soforth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devices,and materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications and patents mentioned herein are incorporated herein byreference for the purpose of describing and disclosing, for example, theconstructs and methodologies that are described in the publications,which might be used in connection with the presently describedinvention. The publications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the inventors arenot entitled to antedate such disclosure by virtue of prior invention orfor any other reason.

The term “substantially purified” refers to a FGF-21 polypeptide thatmay be substantially or essentially free of components that normallyaccompany or interact with the protein as found in its naturallyoccurring environment, i.e. a native cell, or host cell in the case ofrecombinantly produced FGF-21 polypeptides. FGF-21 polypeptide that maybe substantially free of cellular material includes preparations ofprotein having less than about 30%, less than about 25%, less than about20%, less than about 15%, less than about 10%, less than about 5%, lessthan about 4%, less than about 3%, less than about 2%, or less thanabout 1% (by dry weight) of contaminating protein. When the FGF-21polypeptide or variant thereof is recombinantly produced by the hostcells, the protein may be present at about 30%, about 25%, about 20%,about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about1% or less of the dry weight of the cells. When the FGF-21 polypeptideor variant thereof is recombinantly produced by the host cells, theprotein may be present in the culture medium at about 5 g/L, about 4g/L, about 3 g/L, about 2 g/L, about 1 g/L, about 750 mg/L, about 500mg/L, about 250 mg/L, about 100 mg/L, about 50 mg/L, about 10 mg/L, orabout 1 mg/L or less of the dry weight of the cells. Thus,“substantially purified” FGF-21 polypeptide as produced by the methodsof the present invention may have a purity level of at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, specifically, a purity level of at least about 75%,80%, 85%, and more specifically, a purity level of at least about 90%, apurity level of at least about 95%, a purity level of at least about 99%or greater as determined by appropriate methods such as SDS/PAGEanalysis, RP-HPLC, SEC, and capillary electrophoresis.

A “recombinant host cell” or “host cell” refers to a cell that includesan exogenous polynucleotide, regardless of the method used forinsertion, for example, direct uptake, transduction, f-mating, or othermethods known in the art to create recombinant host cells. The exogenouspolynucleotide may be maintained as a nonintegrated vector, for example,a plasmid, or alternatively, may be integrated into the host genome.

As used herein, the term “medium” or “media” includes any culturemedium, solution, solid, semi-solid, or rigid support that may supportor contain any host cell, including bacterial host cells, yeast hostcells, insect host cells, plant host cells, eukaryotic host cells,mammalian host cells, CHO cells, prokaryotic host cells, E. coli, orPseudomonas host cells, and cell contents. Thus, the term may encompassmedium in which the host cell has been grown, e.g., medium into whichthe FGF-21 polypeptide has been secreted, including medium either beforeor after a proliferation step. The term also may encompass buffers orreagents that contain host cell lysates, such as in the case where theFGF-21 polypeptide is produced intracellularly and the host cells arelysed or disrupted to release the FGF-21 polypeptide.

“Reducing agent,” as used herein with respect to protein refolding, isdefined as any compound or material which maintains sulfhydryl groups inthe reduced state and reduces intra- or intermolecular disulfide bonds.Suitable reducing agents include, but are not limited to, dithiothreitol(DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine(2-aminoethanethiol), and reduced glutathione. It is readily apparent tothose of ordinary skill in the art that a wide variety of reducingagents are suitable for use in the methods and compositions of thepresent invention.

“Oxidizing agent,” as used hereinwith respect to protein refolding, isdefined as any compound or material which is capable of removing anelectron from a compound being oxidized. Suitable oxidizing agentsinclude, but are not limited to, oxidized glutathione, cystine,cystamine, oxidized dithiothreitol, oxidized erythreitol, and oxygen. Itis readily apparent to those of ordinary skill in the art that a widevariety of oxidizing agents are suitable for use in the methods of thepresent invention.

The term “anti-diabetic agent” shall mean any drug that is useful intreating, preventing, or otherwise reducing the severity of any glucosemetabolism disorder, or any complications thereof, including any of theconditions, disease, or complications described herein. Anti-diabeticagents include insulin, thiazolidinediones, sulfonylureas, benzoic acidderivatives, alpha-glucosidase inhibitors, or the like. Other generalcategories of anti-diabetic agents which may be part of a subjectcomposition include (with defined terms being in quotation marks): “drugarticles” recognized in the official United States Pharmacopoeia orofficial National Formulary (or any supplement thereto); “new drug” and“new animal drug” approved by the FDA of the U.S. as those terms areused in Title 21 of the United States Code; any drug that requiresapproval of a government entity, in the U.S. or abroad (“approveddrug”); any drug that it is necessary to obtain regulatory approval soas to comply with 21 U.S.C. § 355(a) (“regulatory approved drug”); anyagent that is or was subject to a human drug application under 21 U.S.C.§ 379(g) (“human drug”). (All references to statutory code for thisdefinition refer to such code as of the original filing date of thisapplication.) Other anti-diabetic agents are disclosed herein, and areknown to those of skill in the art. It is preferred that the inventiveantidiabetic compositions, as used herein, are capable of reducing HbA1clevels by at least a 10% change from the baseline, and it is moreparticularly preferred that the inventive anti-diabetic compositions, asused herein, are capable of reducing HbA1c levels by at least a 50%change from the baseline. Antidiabetic agents include insulinpotentiators, such as including but not limited to, small moleculeinsulin potentiators, Taurine, Alpha Lipoic Acid, an extract ofMulberry, Chromium, Glutamine, Enicostemma littorale Blume, Scopariadulcis, an extract of Tarragon, Andrographis paniculata, Isomalt,Trehalose or D-Mannose which may further potentiate the secretion oractivity of insulin.

“Denaturing agent” or “denaturant,” as used herein, is defined as anycompound or material which will cause a reversible unfolding of aprotein. The strength of a denaturing agent or denaturant will bedetermined both by the properties and the concentration of theparticular denaturing agent or denaturant. Suitable denaturing agents ordenaturants may be chaotropes, detergents, organic solvents, watermiscible solvents, phospholipids, or a combination of two or more suchagents. Suitable chaotropes include, but are not limited to, urea,guanidine, and sodium thiocyanate. Useful detergents may include, butare not limited to, strong detergents such as sodium dodecyl sulfate, orpolyoxyethylene ethers (e.g. Tween or Triton detergents), Sarkosyl, mildnon-ionic detergents (e.g., digitonin), mild cationic detergents such asN->2,3-(Dioleyoxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents(e.g. sodium cholate or sodium deoxycholate) or zwitterionic detergentsincluding, but not limited to, sulfobetaines (Zwittergent),3-(3-chlolamidopropyl)dimethylammonio-1-propane sulfate (CHAPS), and3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy-1-propane sulfonate(CHAPSO). Organic, water miscible solvents such as acetonitrile, loweralkanols (especially C₂-C₄ alkanols such as ethanol or isopropanol), orlower alkandiols (especially C₂-C₄ alkandiols such as ethylene-glycol)may be used as denaturants. Phospholipids useful in the presentinvention may be naturally occurring phospholipids such asphosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, andphosphatidylinositol or synthetic phospholipid derivatives or variantssuch as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

“Refolding,” as used herein describes any process, reaction or methodwhich transforms disulfide bond containing polypeptides from animproperly folded or unfolded state to a native or properly foldedconformation with respect to disulfide bonds.

“Cofolding,” as used herein, refers specifically to refolding processes,reactions, or methods which employ at least two polypeptides whichinteract with each other and result in the transformation of unfolded orimproperly folded polypeptides to native, properly folded polypeptides.

As used herein, “FGF-21 polypeptide,” “fibroblast growth factor 21” or“FGF-21” and unhyphenated forms thereof shall include those polypeptidesand proteins that have at least one biological activity of a fibroblastgrowth factor 21, as well as FGF-21 analogs, FGF-21 isoforms, FGF-21mimetics, FGF-21 fragments, hybrid FGF-21 proteins, fusion proteins,oligomers and multimers, homologues, glycosylation pattern variants,variants, splice variants, and muteins, thereof, regardless of thebiological activity of same, and further regardless of the method ofsynthesis or manufacture thereof including, but not limited to,recombinant (whether produced from cDNA, genomic DNA, synthetic DNA orother form of nucleic acid), in vitro, in vivo, by microinjection ofnucleic acid molecules, synthetic, transgenic, and gene activatedmethods. The term “FGF-21 polypeptide” and “FGF-21” encompass FGF-21polypeptides comprising one or more amino acid substitutions, additionsor deletions.

Substitutions in a wide variety of amino acid positions innaturally-occurring FGF-21 have been described. Substitutions includingbut not limited to, those that modulate pharmaceutical stability,increase agonist activity, increase protease resistance, convert thepolypeptide into an antagonist, etc. and are encompassed by the term“FGF-21 polypeptide” or “FGF-21.”

For sequences of FGF-21 that lack a leader sequence, see SEQ ID NO: 1,SEQ ID NO: 2 and SEQ ID NO: 5 herein. For sequences of FGF-21 with aleader sequence, see SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ IDNO: 7 herein. In some embodiments, FGF-21 polypeptides of the inventionare substantially identical to SEQ ID NOs: 1-7 or any other sequence ofa FGF-21 polypeptide. Multiple polymorphisms of FGF-21 have beenidentified. Leucine or proline have been described at the same positionin U.S. Patent Publication No. 20010012628 and U.S. Pat. No. 6,716,626.N-terminal leader or signal sequences that differ by 1 amino acid(leucine) are shown in U.S. Pat. No. 6,716,626 and U.S. PatentPublication No. 20040259780. Nucleic acid molecules encoding FGF-21 andFGF-21 polypeptides including mutants and methods to express and purifyFGF-21 polypeptides are well known and include, but are not limited to,those disclosed in U.S. Pat. No. 6,716,626; U.S. Patent Publication Nos.2005/0176631, 2005/0037457, 2004/0185494, 2004/0259780, 2002/0164713,and 2001/0012628; WO 01/36640; WO 03/011213; WO 03/059270; WO 04/110472;WO 05/061712; WO 05/072769; WO 05/091944; WO 05/113606; WO 06/028595; WO06/028714; WO 06/050247; WO 06/065582; WO 06/078463, which areincorporated by reference in their entirety herein.

The term “FGF-21 polypeptide” also includes the pharmaceuticallyacceptable salts and prodrugs, and prodrugs of the salts, polymorphs,hydrates, solvates, biologically-active fragments, biologically activevariants and stereoisomers of the naturally-occurring FGF-21 as well asagonist, mimetic, and antagonist variants of the naturally-occurringFGF-21 and polypeptide fusions thereof. Fusions comprising additionalamino acids at the amino terminus, carboxyl terminus, or both, areencompassed by the term “FGF-21 polypeptide.” Exemplary fusions include,but are not limited to, e.g., methionyl FGF-21 in which a methionine islinked to the N-terminus of FGF-21 resulting from the recombinantexpression of the mature form of FGF-21 lacking the leader or signalpeptide or portion thereof (a methionine is linked to the N-terminus ofFGF-21 resulting from the recombinant expression), fusions for thepurpose of purification (including, but not limited to, topoly-histidine or affinity epitopes), fusions with serum albumin bindingpeptides and fusions with serum proteins such as serum albumin. U.S.Pat. No. 5,750,373, which is incorporated by reference herein, describesa method for selecting novel proteins such as growth hormone andantibody fragment variants having altered binding properties for theirrespective receptor molecules. The method comprises fusing a geneencoding a protein of interest to the carboxy terminal domain of thegene III coat protein of the filamentous phage M13. Chimeric moleculescomprising FGF-21 and one or more other molecules, including but notlimited to, keratinocyte growth factor (KGF) may be generated(Reich-Slotky, R. et al., J. Biol. Chem. 270:29813-29818 (1995)). Thechimeric molecule can contain specific regions or fragments of one orboth of the FGF-21 and KGF molecules. Any such fragments can be preparedfrom the proteins by standard biochemical methods, or by expressing apolynucleotide encoding the fragment. FGF-21, or a fragment thereof, canbe produced as a fusion protein comprising human serum albumin (HSA) ora portion thereof. Such fusion constructs are suitable for enhancingexpression of the FGF-21, or fragment thereof, in an eukaryotic hostcell. Exemplary HSA portions include the N-terminal polypeptide (aminoacids 1-369, 1-419, and intermediate lengths starting with amino acid1), as disclosed in U.S. Pat. No. 5,766,883, and PCT publication WO97/24445, which is incorporated by reference herein. Other chimericpolypeptides can include a HSA protein with FGF-21, or fragmentsthereof, attached to each of the C-terminal and N-terminal ends of theHSA. Such HSA constructs are disclosed in U.S. Pat. No. 5,876,969, whichis incorporated by reference herein. Mammalian cell expression of FGF-21is described in WO 2005/091944 which is incorporated by referenceherein.

Various references disclose modification of polypeptides by polymerconjugation or glycosylation. The term “FGF-21 polypeptide” includespolypeptides conjugated to a polymer such as PEG and may be comprised ofone or more additional derivitizations of cysteine, lysine, or otherresidues. In addition, the FGF-21 polypeptide may comprise a linker orpolymer, wherein the amino acid to which the linker or polymer isconjugated may be a non-natural amino acid according to the presentinvention, or may be conjugated to a naturally encoded amino acidutilizing techniques known in the art such as coupling to lysine orcysteine.

Polymer conjugation of FGF-21 and other polypeptides has been reported.See, e.g. WO 2005/091944 which is incorporated by reference herein. U.S.Pat. No. 4,904,584 discloses PEGylated lysine depleted polypeptides,wherein at least one lysine residue has been deleted or replaced withany other amino acid residue. WO 99/67291 discloses a process forconjugating a protein with PEG, wherein at least one amino acid residueon the protein is deleted and the protein is contacted with PEG underconditions sufficient to achieve conjugation to the protein. WO 99/03887discloses PEGylated variants of polypeptides belonging to the growthhormone superfamily, wherein a cysteine residue has been substitutedwith a non-essential amino acid residue located in a specified region ofthe polypeptide. WO 00/26354 discloses a method of producing aglycosylated polypeptide variant with reduced allergenicity, which ascompared to a corresponding parent polypeptide comprises at least oneadditional glycosylation site. U.S. Pat. No. 5,218,092, which isincorporated by reference herein, discloses modification of granulocytecolony stimulating factor (G-CSF) and other polypeptides so as tointroduce at least one additional carbohydrate chain as compared to thenative polypeptide.

The term “FGF-21 polypeptide” also includes glycosylated FGF-21, such asbut not limited to, polypeptides glycosylated at any amino acidposition, N-linked or O-linked glycosylated forms of the polypeptide.Variants containing single nucleotide changes are also considered asbiologically active variants of FGF-21 polypeptide. In addition, splicevariants are also included. The term “FGF-21 polypeptide” also includesFGF-21 polypeptide heterodimers, homodimers, heteromultimers, orhomomultimers of any one or more FGF-21 polypeptides or any otherpolypeptide, protein, carbohydrate, polymer, small molecule, linker,ligand, or other biologically active molecule of any type, linked bychemical means or expressed as a fusion protein, as well as polypeptideanalogues containing, for example, specific deletions or othermodifications yet maintain biological activity.

All references to amino acid positions in FGF-21 described herein arebased on the position in SEQ ID NO: 1, unless otherwise specified (i.e.,when it is stated that the comparison is based on SEQ ID NO: 2, 3, 4, 5,6, 7, or other FGF-21 sequence). For example, the amino acid at position77 of SEQ ID NO: 1, is an arginine and the corresponding arginine islocated in SEQ ID NO: 2 at position 87. Those of skill in the art willappreciate that amino acid positions corresponding to positions in SEQID NO: 1 can be readily identified in any other FGF-21 molecule such asSEQ ID NO: 2, 3, 4, 5, 6, and 7. Those of skill in the art willappreciate that amino acid positions corresponding to positions in SEQID NO: 1, 2, 3, 4, 5, 6, 7 or any other FGF-21 sequence can be readilyidentified in any other FGF-21 molecule such as FGF-21 fusions,variants, fragments, etc. For example, sequence alignment programs suchas BLAST can be used to align and identify a particular position in aprotein that corresponds with a position in SEQ ID NO: 1, 2, 3, 4, 5, 6,7 or other FGF-21 sequence. Substitutions, deletions or additions ofamino acids described herein in reference to SEQ ID NO: 1, 2, 3, 4, 5,6, 7, or other FGF-21 sequence are intended to also refer tosubstitutions, deletions or additions in corresponding positions inFGF-21 fusions, variants, fragments, etc. described herein or known inthe art and are expressly encompassed by the present invention.

The term “FGF-21 polypeptide” or “FGF-21” encompasses FGF-21polypeptides comprising one or more amino acid substitutions, additionsor deletions. FGF-21 polypeptides of the present invention may becomprised of modifications with one or more natural amino acids inconjunction with one or more non-natural amino acid modification.Exemplary substitutions in a wide variety of amino acid positions innaturally-occurring FGF-21 polypeptides have been described, includingbut not limited to substitutions that modulate pharmaceutical stability,that modulate one or more of the biological activities of the FGF-21polypeptide, such as but not limited to, increase agonist activity,increase solubility of the polypeptide, decrease proteasesusceptibility, convert the polypeptide into an antagonist, etc. and areencompassed by the term “FGF-21 polypeptide.” In some embodiments, theFGF-21 antagonist comprises a non-naturally encoded amino acid linked toa water soluble polymer that is present in a receptor binding region ofthe FGF-21 molecule.

In some embodiments, the FGF-21 polypeptides further comprise anaddition, substitution or deletion that modulates biological activity ofthe FGF-21 polypeptide. For example, the additions, substitutions ordeletions may modulate one or more properties or activities of FGF-21.For example, the additions, substitutions or deletions may modulateaffinity for the FGF-21 polypeptide receptor, modulate circulatinghalf-life, modulate therapeutic half-life, modulate stability of thepolypeptide, modulate cleavage by proteases, modulate dose, modulaterelease or bio-availability, facilitate purification, or improve oralter a particular route of administration. Similarly, FGF-21polypeptides may comprise protease cleavage sequences, reactive groups,antibody-binding domains (including but not limited to, FLAG orpoly-His) or other affinity based sequences (including but not limitedto, FLAG, poly-His, GST, etc.) or linked molecules (including but notlimited to, biotin) that improve detection (including but not limitedto, GFP), purification or other traits of the polypeptide.

The term “FGF-21 polypeptide” also encompasses homodimers, heterodimers,homomultimers, and heteromultimers that are linked, including but notlimited to those linked directly via non-naturally encoded amino acidside chains, either to the same or different non-naturally encoded aminoacid side chains, to naturally-encoded amino acid side chains, orindirectly via a linker. Exemplary linkers including but are not limitedto, small organic compounds, water soluble polymers of a variety oflengths such as poly(ethylene glycol) or polydextran, or polypeptides ofvarious lengths.

A “non-naturally encoded amino acid” refers to an amino acid that is notone of the 20 common amino acids or pyrrolysine or selenocysteine. Otherterms that may be used synonymously with the term “non-naturally encodedamino acid” are “non-natural amino acid,” “unnatural amino acid,”“non-naturally-occurring amino acid,” and variously hyphenated andnon-hyphenated versions thereof. The term “non-naturally encoded aminoacid” also includes, but is not limited to, amino acids that occur bymodification (e.g. post-translational modifications) of a naturallyencoded amino acid (including but not limited to, the 20 common aminoacids or pyrrolysine and selenocysteine) but are not themselvesnaturally incorporated into a growing polypeptide chain by thetranslation complex. Examples of such non-naturally-occurring aminoacids include, but are not limited to, N-acetylglucosaminyl-L-serine,N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

An “amino terminus modification group” refers to any molecule that canbe attached to the amino terminus of a polypeptide. Similarly, a“carboxy terminus modification group” refers to any molecule that can beattached to the carboxy terminus of a polypeptide. Terminus modificationgroups include, but are not limited to, various water soluble polymers,peptides or proteins such as serum albumin, or other moieties thatincrease serum half-life of peptides.

The terms “functional group”, “active moiety”, “activating group”,“leaving group”, “reactive site”, “chemically reactive group” and“chemically reactive moiety” are used in the art and herein to refer todistinct, definable portions or units of a molecule. The terms aresomewhat synonymous in the chemical arts and are used herein to indicatethe portions of molecules that perform some function or activity and arereactive with other molecules.

The term “linkage” or “linker” is used herein to refer to groups orbonds that normally are formed as the result of a chemical reaction andtypically are covalent linkages. Hydrolytically stable linkages meansthat the linkages are substantially stable in water and do not reactwith water at useful pH values, including but not limited to, underphysiological conditions for an extended period of time, perhaps evenindefinitely. Hydrolytically unstable or degradable linkages mean thatthe linkages are degradable in water or in aqueous solutions, includingfor example, blood. Enzymatically unstable or degradable linkages meanthat the linkage can be degraded by one or more enzymes. As understoodin the art, PEG and related polymers may include degradable linkages inthe polymer backbone or in the linker group between the polymer backboneand one or more of the terminal functional groups of the polymermolecule. For example, ester linkages formed by the reaction of PEGcarboxylic acids or activated PEG carboxylic acids with alcohol groupson a biologically active agent generally hydrolyze under physiologicalconditions to release the agent. Other hydrolytically degradablelinkages include, but are not limited to, carbonate linkages; iminelinkages resulted from reaction of an amine and an aldehyde; phosphateester linkages formed by reacting an alcohol with a phosphate group;hydrazone linkages which are reaction product of a hydrazide and analdehyde; acetal linkages that are the reaction product of an aldehydeand an alcohol; orthoester linkages that are the reaction product of aformate and an alcohol; peptide linkages formed by an amine group,including but not limited to, at an end of a polymer such as PEG, and acarboxyl group of a peptide; and oligonucleotide linkages formed by aphosphoramidite group, including but not limited to, at the end of apolymer, and a 5′ hydroxyl group of an oligonucleotide.

The term “biologically active molecule”, “biologically active moiety” or“biologically active agent” when used herein means any substance whichcan affect any physical or biochemical properties of a biologicalsystem, pathway, molecule, or interaction relating to an organism,including but not limited to, viruses, bacteria, bacteriophage,transposon, prion, insects, fungi, plants, animals, and humans. Inparticular, as used herein, biologically active molecules include, butare not limited to, any substance intended for diagnosis, cure,mitigation, treatment, or prevention of disease in humans or otheranimals, or to otherwise enhance physical or mental well-being of humansor animals. Examples of biologically active molecules include, but arenot limited to, peptides, proteins, enzymes, small molecule drugs,vaccines, immunogens, hard drugs, soft drugs, carbohydrates, inorganicatoms or molecules, dyes, lipids, nucleosides, radionuclides,oligonucleotides, toxoids, toxins, prokaryotic and eukaryotic cells,viruses, polysaccharides, nucleic acids and portions thereof obtained orderived from viruses, bacteria, insects, animals or any other cell orcell type, liposomes, microparticles and micelles. Classes ofbiologically active agents that are suitable for use with the inventioninclude, but are not limited to, drugs, prodrugs, radionuclides, imagingagents, polymers, antibiotics, fungicides, anti-viral agents,anti-inflammatory agents, anti-tumor agents, cardiovascular agents,anti-anxiety agents, hormones, growth factors, steroidal agents,microbially derived toxins, and the like.

A “bifunctional polymer” refers to a polymer comprising two discretefunctional groups that are capable of reacting specifically with othermoieties (including but not limited to, amino acid side groups) to formcovalent or non-covalent linkages. A bifunctional linker having onefunctional group reactive with a group on a particular biologicallyactive component, and another group reactive with a group on a secondbiological component, may be used to form a conjugate that includes thefirst biologically active component, the bifunctional linker and thesecond biologically active component. Many procedures and linkermolecules for attachment of various compounds to peptides are known.See, e.g., European Patent Publication No. 188,256; U.S. Pat. Nos.4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; and 4,569,789which are incorporated by reference herein. A “multi-functional polymer”refers to a polymer comprising two or more discrete functional groupsthat are capable of reacting specifically with other moieties (includingbut not limited to, amino acid side groups) to form covalent ornon-covalent linkages. A bi-functional polymer or multi-functionalpolymer may be any desired length or molecular weight, and may beselected to provide a particular desired spacing or conformation betweenone or more molecules linked to the FGF-21 and its receptor or FGF-21.

Where substituent groups are specified by their conventional chemicalformulas, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, for example, the structure —CH₂O— isequivalent to the structure —OCH₂—.

The term “substituents” includes but is not limited to “non-interferingsubstituents”. “Non-interfering substituents” are those groups thatyield stable compounds. Suitable non-interfering substituents orradicals include, but are not limited to, halo, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ alkoxy, C₁-C₁₂ aralkyl, C₁-C₁₂ alkaryl,C₃-C₁₂ cycloalkyl, C₃-C₁₂ cycloalkenyl, phenyl, substituted phenyl,toluoyl, xylenyl, biphenyl, C₂-C₁₂ alkoxyalkyl, C₂-C₁₂ alkoxyaryl,C₇-C₁₂ aryloxyalkyl, C₇-C₁₂ oxyaryl, C₁-C₆ alkylsulfinyl, C₁-C₁₀alkylsulfonyl, —(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein m is from 1 to 8,aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclicradical, substituted heterocyclic radical, nitroalkyl, —NO₂, —CN,—NRC(O)—(C₁-C₁₀ alkyl), —C(O)—(C₁-C₁₀ alkyl), C₂-C₁₀ alkyl thioalkyl,—C(O)O—(C₁-C₁₀ alkyl), —OH, —SO₂, ═S, —COOH, —NR₂, carbonyl,—C(O)—(C₁-C₁₀ alkyl)-CF₃, —C(O)—CF₃, —C(O)NR₂, —(C₁-C₁₀ aryl)-S—(C₆-C₁₀aryl), —C(O)—(C₁-C₁₀ aryl), —(CH₂)_(m)—O—(—(CH₂)_(m)—O—(C₁-C₁₀ alkyl)wherein each m is from 1 to 8, —C(O)NR₂, —C(S)NR₂, SO₂NR₂, —NRC(O) NR₂,—NRC(S) NR₂, salts thereof, and the like. Each R as used herein is H,alkyl or substituted alkyl, aryl or substituted aryl, aralkyl, oralkaryl.

The term “halogen” includes fluorine, chlorine, iodine, and bromine.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.” Alkyl groups whichare limited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by the structures —CH₂CH₂—and —CH₂CH₂CH₂CH₂—, and furtherincludes those groups described below as “heteroalkylene.” Typically, analkyl (or alkylene) group will have from 1 to 24 carbon atoms, withthose groups having 10 or fewer carbon atoms being a particularembodiment of the methods and compositions described herein. A “loweralkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂-CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, the same or different heteroatoms can also occupyeither or both of the chain termini (including but not limited to,alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino,aminooxyalkylene, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′—and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Thus, a cycloalkylor heterocycloalkyl include saturated, partially unsaturated and fullyunsaturated ring linkages. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Examples of cycloalkyl include, butare not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl,3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkylinclude, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl),1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,2-piperazinyl, and the like. Additionally, the term encompasses bicyclicand tricyclic ring structures. Similarly, the term “heterocycloalkylene”by itself or as part of another substituent means a divalent radicalderived from heterocycloalkyl, and the term “cycloalkylene” by itself oras part of another substituent means a divalent radical derived fromcycloalkyl.

As used herein, the term “water soluble polymer” refers to any polymerthat is soluble in aqueous solvents. Linkage of water soluble polymersto FGF-21 polypeptides can result in changes including, but not limitedto, increased or modulated serum half-life, or increased or modulatedtherapeutic half-life relative to the unmodified form, modulatedimmunogenicity, modulated physical association characteristics such asaggregation and multimer formation, altered receptor binding, alteredbinding to one or more binding partners, and altered receptordimerization or multimerization. The water soluble polymer may or maynot have its own biological activity, and may be utilized as a linkerfor attaching FGF-21 to other substances, including but not limited toone or more FGF-21 polypeptides, or one or more biologically activemolecules. Suitable polymers include, but are not limited to,polyethylene glycol, polyethylene glycol propionaldehyde, mono C1-C10alkoxy or aryloxy derivatives thereof (described in U.S. Pat. No.5,252,714 which is incorporated by reference herein),monomethoxy-polyethylene glycol, polyvinyl pyrrolidone, polyvinylalcohol, polyamino acids, divinylether maleic anhydride,N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivativesincluding dextran sulfate, polypropylene glycol, polypropyleneoxide/ethylene oxide copolymer, polyoxyethylated polyol, heparin,heparin fragments, polysaccharides, oligosaccharides, glycans, celluloseand cellulose derivatives, including but not limited to methylcelluloseand carboxymethyl cellulose, starch and starch derivatives,polypeptides, polyalkylene glycol and derivatives thereof, copolymers ofpolyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers,and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, and the like, ormixtures thereof. Examples of such water soluble polymers include, butare not limited to, polyethylene glycol and serum albumin.

As used herein, the term “polyalkylene glycol” or “poly(alkene glycol)”refers to polyethylene glycol (poly(ethylene glycol)), polypropyleneglycol, polybutylene glycol, and derivatives thereof. The term“polyalkylene glycol” encompasses both linear and branched polymers andaverage molecular weights of between 0.1 kDa and 100 kDa. Otherexemplary embodiments are listed, for example, in commercial suppliercatalogs, such as Shearwater Corporation's catalog “Polyethylene Glycoland Derivatives for Biomedical Applications” (2001).

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent which can be a single ring or multiplerings (including but not limited to, from 1 to 3 rings) which are fusedtogether or linked covalently. The term “heteroaryl” refers to arylgroups (or rings) that contain from one to four heteroatoms selectedfrom N, O, and S, wherein the nitrogen and sulfur atoms are optionallyoxidized, and the nitrogen atom(s) are optionally quaternized. Aheteroaryl group can be attached to the remainder of the moleculethrough a heteroatom. Non-limiting examples of aryl and heteroarylgroups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl,2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl,pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl,3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl,purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituentsfor each of the above noted aryl and heteroaryl ring systems areselected from the group of acceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(including but not limited to, aryloxy, arylthioxy, arylalkyl) includesboth aryl and heteroaryl rings as defined above. Thus, the term“arylalkyl” is meant to include those radicals in which an aryl group isattached to an alkyl group (including but not limited to, benzyl,phenethyl, pyridylmethyl and the like) including those alkyl groups inwhich a carbon atom (including but not limited to, a methylene group)has been replaced by, for example, an oxygen atom (including but notlimited to, phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl,and the like).

Each of the above terms (including but not limited to, “alkyl,”“heteroalkyl,” “aryl” and “heteroaryl”) are meant to include bothsubstituted and unsubstituted forms of the indicated radical. Exemplarysubstituents for each type of radical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2 m′+1), where m′ is the totalnumber of carbon atoms in such a radical. R′, R″, R′″ and R″″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R″″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF₃ and —CH₂CF₃) and acyl (including but not limited to,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, but are not limited to: halogen, —OR′, ═O, ═NR′, ═N—OR′,—NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R″″, —NR″C(O)₂R′,—NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″,—NRSO₂R′, —CN and —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, andfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number ofopen valences on the aromatic ring system; and where R′, R″, R′″ and R″″are independently selected from hydrogen, alkyl, heteroalkyl, aryl andheteroaryl. When a compound of the invention includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R′″ and R″″ groups when more than one of these groupsis present.

As used herein, the term “modulated serum half-life” means the positiveor negative change in circulating half-life of a modified FGF-21relative to its non-modified form. Serum half-life is measured by takingblood samples at various time points after administration of FGF-21, anddetermining the concentration of that molecule in each sample.Correlation of the serum concentration with time allows calculation ofthe serum half-life. Increased serum half-life desirably has at leastabout two-fold, but a smaller increase may be useful, for example whereit enables a satisfactory dosing regimen or avoids a toxic effect. Insome embodiments, the increase is at least about three-fold, at leastabout five-fold, or at least about ten-fold.

The term “modulated therapeutic half-life” as used herein means thepositive or negative change in the half-life of the therapeuticallyeffective amount of FGF-21, relative to its non-modified form.Therapeutic half-life is measured by measuring pharmacokinetic and/orpharmacodynamic properties of the molecule at various time points afteradministration. Increased therapeutic half-life desirably enables aparticular beneficial dosing regimen, a particular beneficial totaldose, or avoids an undesired effect. In some embodiments, the increasedtherapeutic half-life results from increased potency, increased ordecreased binding of the modified molecule to its target, increased ordecreased breakdown of the molecule by enzymes such as proteases, or anincrease or decrease in another parameter or mechanism of action of thenon-modified molecule or an increase or decrease in receptor-mediatedclearance of the molecule.

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is free of at least some of thecellular components with which it is associated in the natural state, orthat the nucleic acid or protein has been concentrated to a levelgreater than the concentration of its in vivo or in vitro production. Itcan be in a homogeneous state. Isolated substances can be in either adry or semi-dry state, or in solution, including but not limited to, anaqueous solution. It can be a component of a pharmaceutical compositionthat comprises additional pharmaceutically acceptable carriers and/orexcipients. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinwhich is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames which flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to substantially one band in anelectrophoretic gel. Particularly, it may mean that the nucleic acid orprotein is at least 85% pure, at least 90% pure, at least 95% pure, atleast 99% or greater pure.

The term “nucleic acid” refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless specifically limited otherwise,the term also refers to oligonucleotide analogs including PNA(peptidonucleic acid), analogs of DNA used in antisense technology(phosphorothioates, phosphoroamidates, and the like). Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (including but notlimited to, degenerate codon substitutions) and complementary sequencesas well as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers in which one or more amino acid residues is anon-naturally encoded amino acid. As used herein, the terms encompassamino acid chains of any length, including full length proteins, whereinthe amino acid residues are linked by covalent peptide bonds.

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids are the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine) and pyrrolysine and selenocysteine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, such as,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (such as, norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Reference to an amino acidincludes, for example, naturally occurring proteogenic L-amino acids;D-amino acids, chemically modified amino acids such as amino acidvariants and derivatives; naturally occurring non-proteogenic aminoacids such as β-alanine, ornithine, etc.; and chemically synthesizedcompounds having properties known in the art to be characteristic ofamino acids. Examples of non-naturally occurring amino acids include,but are not limited to, α-methyl amino acids (e.g., α-methyl alanine),D-amino acids, histidine-like amino acids (e.g., 2-amino-histidine,β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine andα-methyl-histidine), amino acids having an extra methylene in the sidechain (“homo” amino acids), and amino acids in which a carboxylic acidfunctional group in the side chain is replaced with a sulfonic acidgroup (e.g., cysteic acid).

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of ordinary skill inthe art will recognize that each codon in a nucleic acid (except AUG,which is ordinarily the only codon for methionine, and TGG, which isordinarily the only codon for tryptophan) can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide is implicit in each describedsequence.

As to amino acid sequences, one of ordinary skill in the art willrecognize that individual substitutions, deletions or additions to anucleic acid, peptide, polypeptide, or protein sequence which alters,adds or deletes a single amino acid or a small percentage of amino acidsin the encoded sequence is a “conservatively modified variant” where thealteration results in the deletion of an amino acid, addition of anamino acid, or substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are known to those of ordinary skill in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention.

Conservative substitution tables providing functionally similar aminoacids are known to those of ordinary skill in the art. The followingeight groups each contain amino acids that are conservativesubstitutions for one another:

-   1) Alanine (A), Glycine (G);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);-   7) Serine (S), Threonine (T); and-   8) Cysteine (C), Methionine (M)    (see, e.g., Creighton, Proteins: Structures and Molecular Properties    (W H Freeman & Co.; 2nd edition (December 1993)

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. Sequences are“substantially identical” if they have a percentage of amino acidresidues or nucleotides that are the same (i.e., about 60% identity,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, orabout 95% identity over a specified region), when compared and alignedfor maximum correspondence over a comparison window, or designatedregion as measured using one of the following sequence comparisonalgorithms (or other algorithms available to persons of ordinary skillin the art) or by manual alignment and visual inspection. Thisdefinition also refers to the complement of a test sequence. Theidentity can exist over a region that is at least about 50 amino acidsor nucleotides in length, or over a region that is 75-100 amino acids ornucleotides in length, or, where not specified, across the entiresequence of a polynucleotide or polypeptide. A polynucleotide encoding apolypeptide of the present invention, including homologs from speciesother than human, may be obtained by a process comprising the steps ofscreening a library under stringent hybridization conditions with alabeled probe having a polynucleotide sequence of the invention or afragment thereof, and isolating full-length cDNA and genomic clonescontaining said polynucleotide sequence. Such hybridization techniquesare well known to the skilled artisan.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are known to those of ordinary skill in the art. Optimalalignment of sequences for comparison can be conducted, including butnot limited to, by the local homology algorithm of Smith and Waterman(1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci.USA 85:2444, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection (see, e.g., Ausubel et al., CurrentProtocols in Molecular Biology (1995 supplement)).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1997) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Informationavailable at the World Wide Web at ncbi.nlm.nih.gov. The BLAST algorithmparameters W, T, and X determine the sensitivity and speed of thealignment. The BLASTN program (for nucleotide sequences) uses asdefaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4 anda comparison of both strands. For amino acid sequences, the BLASTPprogram uses as defaults a wordlength of 3, and expectation (E) of 10,and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of10, M=5, N=−4, and a comparison of both strands. The BLAST algorithm istypically performed with the “low complexity” filter turned off.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, or less than about0.01, or less than about 0.001.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (including but not limited to,total cellular or library DNA or RNA).

The phrase “stringent hybridization conditions” refers to hybridizationof sequences of DNA, RNA, PNA, or other nucleic acid mimics, orcombinations thereof under conditions of low ionic strength and hightemperature as is known in the art. Typically, under stringentconditions a probe will hybridize to its target subsequence in a complexmixture of nucleic acid (including but not limited to, total cellular orlibrary DNA or RNA) but does not hybridize to other sequences in thecomplex mixture. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Probes, “Overview of principles of hybridization and thestrategy of nucleic acid assays” (1993). Generally, stringent conditionsare selected to be about 5-10° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength pH. TheT_(m) is the temperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions may be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (including butnot limited to, 10 to 50 nucleotides) and at least about 60° C. for longprobes (including but not limited to, greater than 50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. For selective or specifichybridization, a positive signal may be at least two times background,optionally 10 times background hybridization. Exemplary stringenthybridization conditions can be as following: 50% formamide, 5×SSC, and1% SDS, incubating at 42° C., or 5×SSC, 1% SDS, incubating at 65° C.,with wash in 0.2×SSC, and 0.1% SDS at 65° C. Such washes can beperformed for 5, 15, 30, 60, 120, or more minutes.

As used herein, the term “eukaryote” refers to organisms belonging tothe phylogenetic domain Eucarya such as animals (including but notlimited to, mammals, insects, reptiles, birds, etc.), ciliates, plants(including but not limited to, monocots, dicots, algae, etc.), fungi,yeasts, flagellates, microsporidia, protists, etc.

As used herein, the term “non-eukaryote” refers to non-eukaryoticorganisms. For example, a non-eukaryotic organism can belong to theEubacteria (including but not limited to, Escherichia coli, Thermusthermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens,Pseudomonas aeruginosa, Pseudomonas putida, etc.) phylogenetic domain,or the Archaea (including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium such as Haloferaxvolcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus,Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.)phylogenetic domain.

The term “subject” as used herein, refers to an animal, in someembodiments a mammal, and in other embodiments a human, who is theobject of treatment, observation or experiment. An animal may be acompanion animal (e.g., dogs, cats, and the like), farm animal (e.g.,cows, sheep, pigs, horses, and the like) or a laboratory animal (e.g.,rats, mice, guinea pigs, and the like).

The term “effective amount” as used herein refers to that amount of themodified non-natural amino acid polypeptide being administered whichwill relieve to some extent one or more of the symptoms of the disease,condition or disorder being treated. Compositions containing themodified non-natural amino acid polypeptide described herein can beadministered for prophylactic, enhancing, and/or therapeutic treatments.

The terms “enhance” or “enhancing” means to increase or prolong eitherin potency or duration a desired effect. Thus, in regard to enhancingthe effect of therapeutic agents, the term “enhancing” refers to theability to increase or prolong, either in potency or duration, theeffect of other therapeutic agents on a system. An “enhancing-effectiveamount,” as used herein, refers to an amount adequate to enhance theeffect of another therapeutic agent in a desired system. When used in apatient, amounts effective for this use will depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician.

The term “modified,” as used herein refers to any changes made to agiven polypeptide, such as changes to the length of the polypeptide, theamino acid sequence, chemical structure, co-translational modification,or post-translational modification of a polypeptide. The form“(modified)” term means that the polypeptides being discussed areoptionally modified, that is, the polypeptides under discussion can bemodified or unmodified.

The term “post-translationally modified” refers to any modification of anatural or non-natural amino acid that occurs to such an amino acidafter it has been incorporated into a polypeptide chain. The termencompasses, by way of example only, co-translational in vivomodifications, co-translational in vitro modifications (such as in acell-free translation system), post-translational in vivo modifications,and post-translational in vitro modifications.

In prophylactic applications, compositions containing the FGF-21polypeptide are administered to a patient susceptible to or otherwise atrisk of a particular disease, disorder or condition. Such an amount isdefined to be a “prophylactically effective amount.” In this use, theprecise amounts also depend on the patient's state of health, weight,and the like. It is considered well within the skill of the art for oneto determine such prophylactically effective amounts by routineexperimentation (e.g., a dose escalation clinical trial).

The term “protected” refers to the presence of a “protecting group” ormoiety that prevents reaction of the chemically reactive functionalgroup under certain reaction conditions. The protecting group will varydepending on the type of chemically reactive group being protected. Forexample, if the chemically reactive group is an amine or a hydrazide,the protecting group can be selected from the group oftert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). Ifthe chemically reactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also be usedin or with the methods and compositions described herein, includingphotolabile groups such as Nvoc and MeNvoc. Other protecting groupsknown in the art may also be used in or with the methods andcompositions described herein.

By way of example only, blocking/protecting groups may be selected from:

Other protecting groups are described in Greene and Wuts, ProtectiveGroups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, N.Y.,1999, which is incorporated herein by reference in its entirety.

In therapeutic applications, compositions containing the modifiednon-natural amino acid polypeptide are administered to a patient alreadysuffering from a disease, condition or disorder, in an amount sufficientto cure or at least partially arrest the symptoms of the disease,disorder or condition. Such an amount is defined to be a“therapeutically effective amount,” and will depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician. It is considered well within the skill of theart for one to determine such therapeutically effective amounts byroutine experimentation (e.g., a dose escalation clinical trial).

The term “treating” is used to refer to either prophylactic and/ortherapeutic treatments.

Non-naturally encoded amino acid polypeptides presented herein mayinclude isotopically-labelled compounds with one or more atoms replacedby an atom having an atomic mass or mass number different from theatomic mass or mass number usually found in nature. Examples of isotopesthat can be incorporated into the present compounds include isotopes ofhydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as ²H,³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ³⁶C₁, respectively. Certainisotopically-labelled compounds described herein, for example those intowhich radioactive isotopes such as ³H and ¹⁴C are incorporated, may beuseful in drug and/or substrate tissue distribution assays. Further,substitution with isotopes such as deuterium, i.e., 41, can affordcertain therapeutic advantages resulting from greater metabolicstability, for example increased in vivo half-life or reduced dosagerequirements.

All isomers including but not limited to diastereomers, enantiomers, andmixtures thereof are considered as part of the compositions describedherein. In additional or further embodiments, the non-naturally encodedamino acid polypeptides are metabolized upon administration to anorganism in need to produce a metabolite that is then used to produce adesired effect, including a desired therapeutic effect. In further oradditional embodiments are active metabolites of non-naturally encodedamino acid polypeptides.

In some situations, non-naturally encoded amino acid polypeptides mayexist as tautomers. In addition, the non-naturally encoded amino acidpolypeptides described herein can exist in unsolvated as well assolvated forms with pharmaceutically acceptable solvents such as water,ethanol, and the like. The solvated forms are also considered to bedisclosed herein. Those of ordinary skill in the art will recognize thatsome of the compounds herein can exist in several tautomeric forms. Allsuch tautomeric forms are considered as part of the compositionsdescribed herein.

Unless otherwise indicated, conventional methods of mass spectroscopy,NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniquesand pharmacology, within the skill of the art are employed.

DETAILED DESCRIPTION I. Introduction

FGF-21 molecules comprising at least one unnatural amino acid areprovided in the invention. In certain embodiments of the invention, theFGF-21 polypeptide with at least one unnatural amino acid includes atleast one post-translational modification. In one embodiment, the atleast one post-translational modification comprises attachment of amolecule including but not limited to, a label, a dye, a polymer, awater-soluble polymer, a derivative of polyethylene glycol, aphotocrosslinker, a radionuclide, a cytotoxic compound, a drug, anaffinity label, a photoaffinity label, a reactive compound, a resin, asecond protein or polypeptide or polypeptide analog, an antibody orantibody fragment, a metal chelator, a cofactor, a fatty acid, acarbohydrate, a polynucleotide, a DNA, a RNA, an antisensepolynucleotide, a saccharide, a water-soluble dendrimer, a cyclodextrin,an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spinlabel, a fluorophore, a metal-containing moiety, a radioactive moiety, anovel functional group, a group that covalently or noncovalentlyinteracts with other molecules, a photocaged moiety, an actinicradiation excitable moiety, a photoisomerizable moiety, biotin, aderivative of biotin, a biotin analogue, a moiety incorporating a heavyatom, a chemically cleavable group, a photocleavable group, an elongatedside chain, a carbon-linked sugar, a redox-active agent, an aminothioacid, a toxic moiety, an isotopically labeled moiety, a biophysicalprobe, a phosphorescent group, a chemiluminescent group, an electrondense group, a magnetic group, an intercalating group, a chromophore, anenergy transfer agent, a biologically active agent, a detectable label,a small molecule, a quantum dot, a nanotransmitter, a radionucleotide, aradiotransmitter, a neutron-capture agent, or any combination of theabove or any other desirable compound or substance, comprising a secondreactive group to at least one unnatural amino acid comprising a firstreactive group utilizing chemistry methodology that is known to one ofordinary skill in the art to be suitable for the particular reactivegroups. For example, the first reactive group is an alkynyl moiety(including but not limited to, in the unnatural amino acidp-propargyloxyphenylalanine, where the propargyl group is also sometimesreferred to as an acetylene moiety) and the second reactive group is anazido moiety, and [3+2] cycloaddition chemistry methodologies areutilized. In another example, the first reactive group is the azidomoiety (including but not limited to, in the unnatural amino acidp-azido-L-phenylalanine) and the second reactive group is the alkynylmoiety. In certain embodiments of the modified FGF-21 polypeptide of thepresent invention, at least one unnatural amino acid (including but notlimited to, unnatural amino acid containing a keto functional group)comprising at least one post-translational modification, is used wherethe at least one post-translational modification comprises a saccharidemoiety. In certain embodiments, the post-translational modification ismade in vivo in a eukaryotic cell or in a non-eukaryotic cell. A linker,polymer, water soluble polymer, or other molecule may attach themolecule to the polypeptide. The molecule may be linked directly to thepolypeptide.

In certain embodiments, the protein includes at least onepost-translational modification that is made in vivo by one host cell,where the post-translational modification is not normally made byanother host cell type. In certain embodiments, the protein includes atleast one post-translational modification that is made in vivo by aeukaryotic cell, where the post-translational modification is notnormally made by a non-eukaryotic cell. Examples of post-translationalmodifications include, but are not limited to, glycosylation,acetylation, acylation, lipid-modification, palmitoylation, palmitateaddition, phosphorylation, glycolipid-linkage modification, and thelike. In one embodiment, the post-translational modification comprisesattachment of an oligosaccharide to an asparagine by a GlcNAc-asparaginelinkage (including but not limited to, where the oligosaccharidecomprises (GlcNAc-Man)₂-Man-GlcNAc-GlcNAc, and the like). In anotherembodiment, the post-translational modification comprises attachment ofan oligosaccharide (including but not limited to, Gal-GalNAc,Gal-GlcNAc, etc.) to a serine or threonine by a GalNAc-serine, aGalNAc-threonine, a GlcNAc-serine, or a GlcNAc-threonine linkage. Incertain embodiments, a protein or polypeptide of the invention cancomprise a secretion or localization sequence, an epitope tag, a FLAGtag, a polyhistidine tag, a GST fusion, and/or the like. Examples ofsecretion signal sequences include, but are not limited to, aprokaryotic secretion signal sequence, a eukaryotic secretion signalsequence, a eukaryotic secretion signal sequence 5′-optimized forbacterial expression, a novel secretion signal sequence, pectate lyasesecretion signal sequence, Omp A secretion signal sequence, and a phagesecretion signal sequence. Examples of secretion signal sequences,include, but are not limited to, STII (prokaryotic), Fd Gill and M13(phage), Bg12 (yeast), and the signal sequence bla derived from atransposon. Any such sequence may be modified to provide a desiredresult with the polypeptide, including but not limited to, substitutingone signal sequence with a different signal sequence, substituting aleader sequence with a different leader sequence, etc.

The protein or polypeptide of interest can contain at least one, atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, or ten or more unnaturalamino acids. The unnatural amino acids can be the same or different, forexample, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more differentsites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moredifferent unnatural amino acids. In certain embodiments, at least one,but fewer than all, of a particular amino acid present in a naturallyoccurring version of the protein is substituted with an unnatural aminoacid.

The present invention provides methods and compositions based on FGF-21comprising at least one non-naturally encoded amino acid. Introductionof at least one non-naturally encoded amino acid into FGF-21 can allowfor the application of conjugation chemistries that involve specificchemical reactions, including, but not limited to, with one or morenon-naturally encoded amino acids while not reacting with the commonlyoccurring 20 amino acids. In some embodiments, FGF-21 comprising thenon-naturally encoded amino acid is linked to a water soluble polymer,such as polyethylene glycol (PEG), via the side chain of thenon-naturally encoded amino acid. This invention provides a highlyefficient method for the selective modification of proteins with PEGderivatives, which involves the selective incorporation ofnon-genetically encoded amino acids, including but not limited to, thoseamino acids containing functional groups or substituents not found inthe 20 naturally incorporated amino acids, including but not limited toa ketone, an azide or acetylene moiety, into proteins in response to aselector codon and the subsequent modification of those amino acids witha suitably reactive PEG derivative. Once incorporated, the amino acidside chains can then be modified by utilizing chemistry methodologiesknown to those of ordinary skill in the art to be suitable for theparticular functional groups or substituents present in thenon-naturally encoded amino acid. Known chemistry methodologies of awide variety are suitable for use in the present invention toincorporate a water soluble polymer into the protein. Such methodologiesinclude but are not limited to a Huisgen [3+2] cycloaddition reaction(see, e.g., Padwa, A. in Comprehensive Organic Synthesis, Vol. 4, (1991)Ed. Trost, B. M., Pergamon, Oxford, p. 1069-1109; and, Huisgen, R. in1,3-Dipolar Cycloaddition Chemistry, (1984) Ed. Padwa, A., Wiley, NewYork, p. 1-176) with, including but not limited to, acetylene or azidederivatives, respectively.

Because the Huisgen [3+2] cycloaddition method involves a cycloadditionrather than a nucleophilic substitution reaction, proteins can bemodified with extremely high selectivity. The reaction can be carriedout at room temperature in aqueous conditions with excellentregioselectivity (1,4 >1,5) by the addition of catalytic amounts ofCu(I) salts to the reaction mixture. See, e.g., Tornoe, et al., (2002)J. Org. Chem. 67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem.Int. Ed. 41:2596-2599; and WO 03/101972. A molecule that can be added toa protein of the invention through a [3+2] cycloaddition includesvirtually any molecule with a suitable functional group or substituentincluding but not limited to an azido or acetylene derivative. Thesemolecules can be added to an unnatural amino acid with an acetylenegroup, including but not limited to, p-propargyloxyphenylalanine, orazido group, including but not limited to p-azido-phenylalanine,respectively.

The five-membered ring that results from the Huisgen [3+2] cycloadditionis not generally reversible in reducing environments and is stableagainst hydrolysis for extended periods in aqueous environments.Consequently, the physical and chemical characteristics of a widevariety of substances can be modified under demanding aqueous conditionswith the active PEG derivatives of the present invention. Even moreimportantly, because the azide and acetylene moieties are specific forone another (and do not, for example, react with any of the 20 common,genetically-encoded amino acids), proteins can be modified in one ormore specific sites with extremely high selectivity.

The invention also provides water soluble and hydrolytically stablederivatives of PEG derivatives and related hydrophilic polymers havingone or more acetylene or azide moieties. The PEG polymer derivativesthat contain acetylene moieties are highly selective for coupling withazide moieties that have been introduced selectively into proteins inresponse to a selector codon. Similarly, PEG polymer derivatives thatcontain azide moieties are highly selective for coupling with acetylenemoieties that have been introduced selectively into proteins in responseto a selector codon.

More specifically, the azide moieties comprise, but are not limited to,alkyl azides, aryl azides and derivatives of these azides. Thederivatives of the alkyl and aryl azides can include other substituentsso long as the acetylene-specific reactivity is maintained. Theacetylene moieties comprise alkyl and aryl acetylenes and derivatives ofeach. The derivatives of the alkyl and aryl acetylenes can include othersubstituents so long as the azide-specific reactivity is maintained.

The present invention provides conjugates of substances having a widevariety of functional groups, substituents or moieties, with othersubstances including but not limited to a label; a dye; a polymer; awater-soluble polymer; a derivative of polyethylene glycol; aphotocrosslinker; a radionuclide; a cytotoxic compound; a drug; anaffinity label; a photoaffinity label; a reactive compound; a resin; asecond protein or polypeptide or polypeptide analog; an antibody orantibody fragment; a metal chelator; a cofactor; a fatty acid; acarbohydrate; a polynucleotide; a DNA; a RNA; an antisensepolynucleotide; a saccharide; a water-soluble dendrimer; a cyclodextrin;an inhibitory ribonucleic acid; a biomaterial; a nanoparticle; a spinlabel; a fluorophore, a metal-containing moiety; a radioactive moiety; anovel functional group; a group that covalently or noncovalentlyinteracts with other molecules; a photocaged moiety; an actinicradiation excitable moiety; a photoisomerizable moiety; biotin; aderivative of biotin; a biotin analogue; a moiety incorporating a heavyatom; a chemically cleavable group; a photocleavable group; an elongatedside chain; a carbon-linked sugar; a redox-active agent; an aminothioacid; a toxic moiety; an isotopically labeled moiety; a biophysicalprobe; a phosphorescent group; a chemiluminescent group; an electrondense group; a magnetic group; an intercalating group; a chromophore; anenergy transfer agent; a biologically active agent; a detectable label;a small molecule; a quantum dot; a nanotransmitter; a radionucleotide; aradiotransmitter; a neutron-capture agent; or any combination of theabove, or any other desirable compound or substance. The presentinvention also includes conjugates of substances having azide oracetylene moieties with PEG polymer derivatives having the correspondingacetylene or azide moieties. For example, a PEG polymer containing anazide moiety can be coupled to a biologically active molecule at aposition in the protein that contains a non-genetically encoded aminoacid bearing an acetylene functionality. The linkage by which the PEGand the biologically active molecule are coupled includes but is notlimited to the Huisgen [3+2] cycloaddition product.

It is well established in the art that PEG can be used to modify thesurfaces of biomaterials (see, e.g., U.S. Pat. No. 6,610,281; Mehvar,R., J. Pharm Pharm Sci., 3(1):125-136 (2000) which are incorporated byreference herein). The invention also includes biomaterials comprising asurface having one or more reactive azide or acetylene sites and one ormore of the azide- or acetylene-containing polymers of the inventioncoupled to the surface via the Huisgen [3+2] cycloaddition linkage.Biomaterials and other substances can also be coupled to the azide- oracetylene-activated polymer derivatives through a linkage other than theazide or acetylene linkage, such as through a linkage comprising acarboxylic acid, amine, alcohol or thiol moiety, to leave the azide oracetylene moiety available for subsequent reactions.

The invention includes a method of synthesizing the azide- andacetylene-containing polymers of the invention. In the case of theazide-containing PEG derivative, the azide can be bonded directly to acarbon atom of the polymer. Alternatively, the azide-containing PEGderivative can be prepared by attaching a linking agent that has theazide moiety at one terminus to a conventional activated polymer so thatthe resulting polymer has the azide moiety at its terminus. In the caseof the acetylene-containing PEG derivative, the acetylene can be bondeddirectly to a carbon atom of the polymer. Alternatively, theacetylene-containing PEG derivative can be prepared by attaching alinking agent that has the acetylene moiety at one terminus to aconventional activated polymer so that the resulting polymer has theacetylene moiety at its terminus.

More specifically, in the case of the azide-containing PEG derivative, awater soluble polymer having at least one active hydroxyl moietyundergoes a reaction to produce a substituted polymer having a morereactive moiety, such as a mesylate, tresylate, tosylate or halogenleaving group, thereon. The preparation and use of PEG derivativescontaining sulfonyl acid halides, halogen atoms and other leaving groupsare known to those of ordinary skill in the art. The resultingsubstituted polymer then undergoes a reaction to substitute for the morereactive moiety an azide moiety at the terminus of the polymer.Alternatively, a water soluble polymer having at least one activenucleophilic or electrophilic moiety undergoes a reaction with a linkingagent that has an azide at one terminus so that a covalent bond isformed between the PEG polymer and the linking agent and the azidemoiety is positioned at the terminus of the polymer. Nucleophilic andelectrophilic moieties, including amines, thiols, hydrazides,hydrazines, alcohols, carboxylates, aldehydes, ketones, thioesters andthe like, are known to those of ordinary skill in the art.

More specifically, in the case of the acetylene-containing PEGderivative, a water soluble polymer having at least one active hydroxylmoiety undergoes a reaction to displace a halogen or other activatedleaving group from a precursor that contains an acetylene moiety.Alternatively, a water soluble polymer having at least one activenucleophilic or electrophilic moiety undergoes a reaction with a linkingagent that has an acetylene at one terminus so that a covalent bond isformed between the PEG polymer and the linking agent and the acetylenemoiety is positioned at the terminus of the polymer. The use of halogenmoieties, activated leaving group, nucleophilic and electrophilicmoieties in the context of organic synthesis and the preparation and useof PEG derivatives is well established to practitioners in the art.

The invention also provides a method for the selective modification ofproteins to add other substances to the modified protein, including butnot limited to water soluble polymers such as PEG and PEG derivativescontaining an azide or acetylene moiety. The azide- andacetylene-containing PEG derivatives can be used to modify theproperties of surfaces and molecules where biocompatibility, stability,solubility and lack of immunogenicity are important, while at the sametime providing a more selective means of attaching the PEG derivativesto proteins than was previously known in the art.

II. Fibroblast Growth Factors

Because of their potent activities for promoting growth, proliferation,survival and differentiation of a wide variety of cells and tissuetypes, FGFs continue to be pursued as therapeutic agents for a number ofdifferent indications, including wound healing, such as musculo-skeletalconditions, for example, bone fractures, ligament and tissue repair,tendonitis, bursitis, etc.; skin conditions, for example, burns, cuts,lacerations, bed sores, slow healing ulcers, etc.; tissue protection,repair, and the induction of angiogenesis during myocardial infarctionand ischemia, in the treatment of neurological conditions, for example,neuro-degenerative disease and stroke, in the treatment of eye disease,including macular degeneration, and the like.

The fibroblast growth factor (FGF) proteins identified to date belong toa family of signaling molecules that regulate growth and differentiationof a variety of cell types. The significance of FGF proteins to humanphysiology and pathology relates in part to their key roles inembryogenesis, in blood vessel development and growth, and in bonegrowth. In vitro experiments have demonstrated a role for FGF inregulating cell growth and division of endothelial cells, vascularsmooth muscle cells, fibroblasts, and cardiac and skeletal myocytes.Other members of the FGF family and their biological roles are describedin Crossley et al., Development 121:439-451 (1995); Ohuchi et al.,Development 124:2235-2244 (1997); Gemel et al., Genomics 35:253-257(1996); and Ghosh et al., Cell Growth and Differentiation 7:1425-1434(1996).

FGF proteins are also significant to human health and disease because ofa role in cancer cell growth. For example, FGF-8 was identified as anandrogen-induced growth factor in breast and prostate cancer cells.(Tanaka et al., FEBS Lett. 363:226-230 (1995) and P.N.A.S. 89:8928-8932(1992)).

The role of FGF in normal development is being elucidated in partthrough studies of FGF receptors. Wilke, T. et al., Dev. Dynam.210:41-52 (1997) found that FGFR1, FGFR2, and FGFR3 transcripts werelocalized to specific regions of the head during embryonic developmentin chickens. The expression pattern correlated with areas affected byhuman FGFR mutations in Crouzon syndrome, a condition of abnormalintramembranous bone formation. Belluardo, N. et al., Jour. Comp. Neur.379:226-246 (1997) studied localization of FGFR 1, 2, and 3 mRNAs in ratbrain, and found cellular specificity in several brain regions.Furthermore, FGFR1 and FGFR2 mRNAs were expressed in astroglial reactivecells after brain lesion, supporting a role of certain FGF's in braindisease and injury. Ozawa, K. et al., Mol. Brain Res. 41:279-288 (1996)reported that FGF1 and FGF-5 expression increased after birth, whereasFGF3, FGF-6, FGF-7, and FGF-8 genes showed higher expression in lateembryonic stages than in postnatal stages. A cofactor, Klotho beta(Klb), may also be involved with signal transduction of FGF-21 and itsreceptor. Klb has been reported to increase the ability of FGFR1 andFGFR4 to bind FGF21. Klb is a single-pass transmembrane protein andalthough the role of the full transmembrane form is unknown, it has beenshown in regards to FGF23 that Klotho enhanced FGF23 binding andincreased phosphorylation of FGF receptor while Klotho beta has beenshown to enhance FGF-21 binding (H. Kurosu, Y. Ogawa, M. Miyoshi, M.Yamamoto, A. Nandi, K. P. Rosenblatt, M. G. Baum, S. Schiavi, M.-C. Hu,0. W. Moe, M. Kuro-o, Regulation of fibroblast growth factor-23signaling by Klotho. J. Biol. Chem. 281, 6120-6123 (2006) incorporatedherein by reference).

Katoh et al. (International Journal of Oncology (2006) 29:163-168)describe the FGF family and phylogenetic analyses of the family members.Katoh et al. also discuss signaling pathway network in thegastrointestinal tract.

Plotnikov et al. (Cell (1999) 98:641-650) describe the crystal structureof FGF2 with FGF receptor 1 (FGFR1) and the 2-fold symmetric dimer thatis formed between two of these complexes. Plotnikov et al. provide amodel for dimerization of the receptor and induction of dimerization byFGF and heparin.

Additional members of the FGF family are likely to be discovered in thefuture. New members of the FGF family can be identified throughcomputer-aided secondary and tertiary structure analyses of thepredicted protein sequences, and by selection techniques designed toidentify molecules that bind to a particular target.

Thus, the description of the FGF family is provided for illustrativepurposes and by way of example only and not as a limit on the scope ofthe methods, compositions, strategies and techniques described herein.Further, reference to FGF-21 in this application is intended to use thegeneric term as an example of any member of the FGF family. Thus, it isunderstood that the modifications and chemistries described herein withreference to FGF-21 polypeptides or protein can be equally applied toany member of the FGF family, including those specifically listedherein.

III. General Recombinant Nucleic Acid Methods for Use with the Invention

In numerous embodiments of the present invention, nucleic acids encodinga FGF-21 polypeptide of interest will be isolated, cloned and oftenaltered using recombinant methods. Such embodiments are used, includingbut not limited to, for protein expression or during the generation ofvariants, derivatives, expression cassettes, or other sequences derivedfrom a FGF-21 polypeptide. In some embodiments, the sequences encodingthe polypeptides of the invention are operably linked to a heterologouspromoter.

A nucleotide sequence encoding an FGF-21 polypeptide comprising anon-naturally encoded amino acid may be synthesized on the basis of theamino acid sequence of the parent polypeptide, including but not limitedto, having the amino acid sequence shown in SEQ ID NO: 1-7 and thenchanging the nucleotide sequence so as to effect introduction (i.e.,incorporation or substitution) or removal (i.e., deletion orsubstitution) of the relevant amino acid residue(s). The nucleotidesequence may be conveniently modified by site-directed mutagenesis inaccordance with conventional methods. Alternatively, the nucleotidesequence may be prepared by chemical synthesis, including but notlimited to, by using an oligonucleotide synthesizer, whereinoligonucleotides are designed based on the amino acid sequence of thedesired polypeptide, and preferably selecting those codons that arefavored in the host cell in which the recombinant polypeptide will beproduced. For example, several small oligonucleotides coding forportions of the desired polypeptide may be synthesized and assembled byPCR, ligation or ligation chain reaction. See, e.g., Barany, et al.,Proc. Natl. Acad. Sci. 88: 189-193 (1991); U.S. Pat. No. 6,521,427 whichare incorporated by reference herein.

This invention utilizes routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

General texts which describe molecular biological techniques includeBerger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger);Sambrook et al., Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989(“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubelet al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 1999) (“Ausubel”)). These texts describe mutagenesis, the use ofvectors, promoters and many other relevant topics related to, includingbut not limited to, the generation of genes or polynucleotides thatinclude selector codons for production of proteins that includeunnatural amino acids, orthogonal tRNAs, orthogonal synthetases, andpairs thereof.

Various types of mutagenesis are used in the invention for a variety ofpurposes, including but not limited to, to produce novel synthetases ortRNAs, to mutate tRNA molecules, to mutate polynucleotides encodingsynthetases, to produce libraries of tRNAs, to produce libraries ofsynthetases, to produce selector codons, to insert selector codons thatencode unnatural amino acids in a protein or polypeptide of interest.They include but are not limited to site-directed, random pointmutagenesis, homologous recombination, DNA shuffling or other recursivemutagenesis methods, chimeric construction, mutagenesis using uracilcontaining templates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, mutagenesis using gappedduplex DNA or the like, PCT-mediated mutagenesis, or any combinationthereof. Additional suitable methods include point mismatch repair,mutagenesis using repair-deficient host strains, restriction-selectionand restriction-purification, deletion mutagenesis, mutagenesis by totalgene synthesis, double-strand break repair, and the like. Mutagenesis,including but not limited to, involving chimeric constructs, are alsoincluded in the present invention. In one embodiment, mutagenesis can beguided by known information of the naturally occurring molecule oraltered or mutated naturally occurring molecule, including but notlimited to, sequence, sequence comparisons, physical properties,secondary, tertiary, or quaternary structure, crystal structure or thelike.

The texts and examples found herein describe these procedures.Additional information is found in the following publications andreferences cited within: Ling et al., Approaches to DNA mutagenesis: anoverview, Anal Biochem. 254(2): 157-178 (1997); Dale et al.,Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod, Methods Mol. Biol. 57:369-374 (1996); Smith, In vitromutagenesis, Ann. Rev. Genet. 19:423-462 (1985); Botstein & Shortle,Strategies and applications of in vitro mutagenesis, Science229:1193-1201 (1985); Carter, Site-directed mutagenesis, Biochem. J.237:1-7 (1986); Kunkel, The efficiency of oligonucleotide directedmutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D.M.J. eds., Springer Verlag, Berlin) (1987); Kunkel, Rapid andefficient site-specific mutagenesis without phenotypic selection, Proc.Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., Rapid andefficient site-specific mutagenesis without phenotypic selection,Methods in Enzymol. 154, 367-382 (1987); Bass et al., Mutant Trprepressors with new DNA-binding specificities, Science 242:240-245(1988); Zoller & Smith, Oligonucleotide-directed mutagenesis usingM13-derived vectors: an efficient and general procedure for theproduction of point mutations in any DNA fragment, Nucleic Acids Res.10:6487-6500 (1982); Zoller & Smith, Oligonucleotide-directedmutagenesis of DNA fragments cloned into M13 vectors, Methods inEnzymol. 100:468-500 (1983); Zoller & Smith, Oligonucleotide-directedmutagenesis: a simple method using two oligonucleotide primers and asingle-stranded DNA template, Methods in Enzymol. 154:329-350 (1987);Taylor et al., The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA, Nucl. Acids Res. 13: 8749-8764(1985); Taylor et al., The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA, Nucl.Acids Res. 13: 8765-8785 (1985); Nakamaye & Eckstein, Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups andits application to oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 14: 9679-9698 (1986); Sayers et al., 5′-3′ Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 16:791-802 (1988); Sayers et al., Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide, (1988) Nucl. AcidsRes. 16: 803-814; Kramer et al., The gapped duplex DNA approach tooligonucleotide-directed mutation construction, Nucl. Acids Res. 12:9441-9456 (1984); Kramer & Fritz Oligonucleotide-directed constructionof mutations via gapped duplex DNA, Methods in Enzymol. 154:350-367(1987); Kramer et al., Improved enzymatic in vitro reactions in thegapped duplex DNA approach to oligonucleotide-directed construction ofmutations, Nucl. Acids Res. 16: 7207 (1988); Fritz et al.,Oligonucleotide-directed construction of mutations: a gapped duplex DNAprocedure without enzymatic reactions in vitro, Nucl. Acids Res. 16:6987-6999 (1988); Kramer et al., Different base/base mismatches arecorrected with different efficiencies by the methyl-directed DNAmismatch-repair system of E. coli, Cell 38:879-887 (1984); Carter etal., Improved oligonucleotide site-directed mutagenesis using M13vectors, Nucl. Acids Res. 13: 4431-4443 (1985); Carter, Improvedoligonucleotide-directed mutagenesis using M13 vectors, Methods inEnzymol. 154: 382-403 (1987); Eghtedarzadeh & Henikoff, Use ofoligonucleotides to generate large deletions, Nucl. Acids Res. 14: 5115(1986); Wells et al., Importance of hydrogen-bond formation instabilizing the transition state of subtilisin, Phil. Trans. R. Soc.Lond. A 317: 415-423 (1986); Nambiar et al., Total synthesis and cloningof a gene coding for the ribonuclease S protein, Science 223: 1299-1301(1984); Sakmar and Khorana, Total synthesis and expression of a gene forthe alpha-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin), Nucl. Acids Res. 14: 6361-6372 (1988); Wells etal., Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites, Gene 34:315-323 (1985); Grundstromet al., Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’gene synthesis, Nucl. Acids Res. 13: 3305-3316 (1985); Mandecki,Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis, Proc. Natl.Acad. Sci. USA, 83:7177-7181 (1986); Arnold, Protein engineering forunusual environments, Current Opinion in Biotechnology 4:450-455 (1993);Sieber, et al., Nature Biotechnology, 19:456-460 (2001); W. P. C.Stemmer, Nature 370, 389-91 (1994); and, I. A. Lorimer, I. Pastan,Nucleic Acids Res. 23, 3067-8 (1995). Additional details on many of theabove methods can be found in Methods in Enzymology Volume 154, whichalso describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Oligonucleotides, e.g., for use in mutagenesis of the present invention,e.g., mutating libraries of synthetases, or altering tRNAs, aretypically synthesized chemically according to the solid phasephosphoramidite triester method described by Beaucage and Caruthers,Tetrahedron Letts. 22(20):1859-1862, (1981) e.g., using an automatedsynthesizer, as described in Needham-VanDevanter et al., Nucleic AcidsRes., 12:6159-6168 (1984).

The invention also relates to eukaryotic host cells, non-eukaryotic hostcells, and organisms for the in vivo incorporation of an unnatural aminoacid via orthogonal tRNA/RS pairs. Host cells are genetically engineered(including but not limited to, transformed, transduced or transfected)with the polynucleotides of the invention or constructs which include apolynucleotide of the invention, including but not limited to, a vectorof the invention, which can be, for example, a cloning vector or anexpression vector. For example, the coding regions for the orthogonaltRNA, the orthogonal tRNA synthetase, and the protein to be derivatizedare operably linked to gene expression control elements that arefunctional in the desired host cell. The vector can be, for example, inthe form of a plasmid, a cosmid, a phage, a bacterium, a virus, a nakedpolynucleotide, or a conjugated polynucleotide. The vectors areintroduced into cells and/or microorganisms by standard methodsincluding electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA 82,5824 (1985)), infection by viral vectors, high velocity ballisticpenetration by small particles with the nucleic acid either within thematrix of small beads or particles, or on the surface (Klein et al.,Nature 327, 70-73 (1987)), and/or the like.

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for such activities as, for example, screeningsteps, activating promoters or selecting transformants. These cells canoptionally be cultured into transgenic organisms. Other usefulreferences, including but not limited to for cell isolation and culture(e.g., for subsequent nucleic acid isolation) include Freshney (1994)Culture of Animal Cells, a Manual of Basic Technique, third edition,Wiley-Liss, New York and the references cited therein; Payne et al.(1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley &Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds.) (1995) PlantCell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds.)The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.

Several well-known methods of introducing target nucleic acids intocells are available, any of which can be used in the invention. Theseinclude: fusion of the recipient cells with bacterial protoplastscontaining the DNA, electroporation, projectile bombardment, andinfection with viral vectors (discussed further, below), etc. Bacterialcells can be used to amplify the number of plasmids containing DNAconstructs of this invention. The bacteria are grown to log phase andthe plasmids within the bacteria can be isolated by a variety of methodsknown in the art (see, for instance, Sambrook). In addition, kits arecommercially available for the purification of plasmids from bacteria,(see, e.g., EasyPrep™, FlexiPrep™, both from Pharmacia Biotech;StrataClean™ from Stratagene; and, QIAprep™ from Qiagen). The isolatedand purified plasmids are then further manipulated to produce otherplasmids, used to transfect cells or incorporated into related vectorsto infect organisms. Typical vectors contain transcription andtranslation terminators, transcription and translation initiationsequences, and promoters useful for regulation of the expression of theparticular target nucleic acid. The vectors optionally comprise genericexpression cassettes containing at least one independent terminatorsequence, sequences permitting replication of the cassette ineukaryotes, or prokaryotes, or both, (including but not limited to,shuttle vectors) and selection markers for both prokaryotic andeukaryotic systems. Vectors are suitable for replication and integrationin prokaryotes, eukaryotes, or both. See, Gillam & Smith, Gene 8:81(1979); Roberts, et al., Nature, 328:731 (1987); Schneider, E., et al.,Protein Expr. Purif. 6(1):10-14 (1995); Ausubel, Sambrook, Berger (allsupra). A catalogue of bacteria and bacteriophages useful for cloning isprovided, e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria andBacteriophage (1992) Gherna et al. (eds) published by the ATCC.Additional basic procedures for sequencing, cloning and other aspects ofmolecular biology and underlying theoretical considerations are alsofound in Watson et al. (1992) Recombinant DNA Second Edition ScientificAmerican Books, NY. In addition, essentially any nucleic acid (andvirtually any labeled nucleic acid, whether standard or non-standard)can be custom or standard ordered from any of a variety of commercialsources, such as the Midland Certified Reagent Company (Midland, Tex.available on the World Wide Web at mcrc.com), The Great American GeneCompany (Ramona, Calif. available on the World Wide Web at genco.com),ExpressGen Inc. (Chicago, Ill. available on the World Wide Web atexpressgen.com), Operon Technologies Inc. (Alameda, Calif.) and manyothers.

Selector Codons

Selector codons of the invention expand the genetic codon framework ofprotein biosynthetic machinery. For example, a selector codon includes,but is not limited to, a unique three base codon, a nonsense codon, suchas a stop codon, including but not limited to, an amber codon (UAG), anochre codon, or an opal codon (UGA), an unnatural codon, a four or morebase codon, a rare codon, or the like. It is readily apparent to thoseof ordinary skill in the art that there is a wide range in the number ofselector codons that can be introduced into a desired gene orpolynucleotide, including but not limited to, one or more, two or more,three or more, 4, 5, 6, 7, 8, 9, 10 or more in a single polynucleotideencoding at least a portion of the FGF-21 polypeptide.

In one embodiment, the methods involve the use of a selector codon thatis a stop codon for the incorporation of one or more unnatural aminoacids in vivo. For example, an O-tRNA is produced that recognizes thestop codon, including but not limited to, UAG, and is aminoacylated byan O—RS with a desired unnatural amino acid. This O-tRNA is notrecognized by the naturally occurring host's aminoacyl-tRNA synthetases.Conventional site-directed mutagenesis can be used to introduce the stopcodon, including but not limited to, TAG, at the site of interest in apolypeptide of interest. See, e.g., Sayers, J. R., et al. (1988), 5′-3′Exonucleases in phosphorothioate-based oligonucleotide-directedmutagenesis. Nucleic Acids Res, 16:791-802. When the O—RS, O-tRNA andthe nucleic acid that encodes the polypeptide of interest are combinedin vivo, the unnatural amino acid is incorporated in response to the UAGcodon to give a polypeptide containing the unnatural amino acid at thespecified position.

The incorporation of unnatural amino acids in vivo can be done withoutsignificant perturbation of the eukaryotic host cell. For example,because the suppression efficiency for the UAG codon depends upon thecompetition between the O-tRNA, including but not limited to, the ambersuppressor tRNA, and a eukaryotic release factor (including but notlimited to, eRF) (which binds to a stop codon and initiates release ofthe growing peptide from the ribosome), the suppression efficiency canbe modulated by, including but not limited to, increasing the expressionlevel of O-tRNA, and/or the suppressor tRNA.

Unnatural amino acids can also be encoded with rare codons. For example,when the arginine concentration in an in vitro protein synthesisreaction is reduced, the rare arginine codon, AGG, has proven to beefficient for insertion of Ala by a synthetic tRNA acylated withalanine. See, e.g., Ma et al., Biochemistry, 32:7939 (1993). In thiscase, the synthetic tRNA competes with the naturally occurring tRNAArg,which exists as a minor species in Escherichia coli. Some organisms donot use all triplet codons. An unassigned codon AGA in Micrococcusluteus has been utilized for insertion of amino acids in an in vitrotranscription/translation extract. See, e.g., Kowal and Oliver, Nucl.Acid. Res., 25:4685 (1997). Components of the present invention can begenerated to use these rare codons in vivo.

Selector codons also comprise extended codons, including but not limitedto, four or more base codons, such as, four, five, six or more basecodons. Examples of four base codons include, but are not limited to,AGGA, CUAG, UAGA, CCCU and the like. Examples of five base codonsinclude, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA, CUACU,UAGGC and the like. A feature of the invention includes using extendedcodons based on frameshift suppression. Four or more base codons caninsert, including but not limited to, one or multiple unnatural aminoacids into the same protein. For example, in the presence of mutatedO-tRNAs, including but not limited to, a special frameshift suppressortRNAs, with anticodon loops, for example, with at least 8-10 ntanticodon loops, the four or more base codon is read as single aminoacid. In other embodiments, the anticodon loops can decode, includingbut not limited to, at least a four-base codon, at least a five-basecodon, or at least a six-base codon or more. Since there are 256possible four-base codons, multiple unnatural amino acids can be encodedin the same cell using a four or more base codon. See, Anderson et al.,(2002) Exploring the Limits of Codon and Anticodon Size, Chemistry andBiology, 9:237-244; Magliery, (2001) Expanding the Genetic Code:Selection of Efficient Suppressors of Four-base Codons andIdentification of “Shifty” Four-base Codons with a Library Approach inEscherichia coli, J. Mol. Biol. 307: 755-769.

For example, four-base codons have been used to incorporate unnaturalamino acids into proteins using in vitro biosynthetic methods. See,e.g., Ma et al., (1993) Biochemistry, 32:7939; and Hohsaka et al.,(1999) J. Am. Chem. Soc., 121:34. CGGG and AGGU were used tosimultaneously incorporate 2-naphthylalanine and an NBD derivative oflysine into streptavidin in vitro with two chemically acylatedframeshift suppressor tRNAs. See, e.g., Hohsaka et al., (1999) J. Am.Chem. Soc., 121:12194. In an in vivo study, Moore et al. examined theability of tRNALeu derivatives with NCUA anticodons to suppress UAGNcodons (N can be U, A, G, or C), and found that the quadruplet UAGA canbe decoded by a tRNALeu with a UCUA anticodon with an efficiency of 13to 26% with little decoding in the 0 or −1 frame. See, Moore et al.,(2000) J. Mol. Biol., 298:195. In one embodiment, extended codons basedon rare codons or nonsense codons can be used in the present invention,which can reduce missense readthrough and frameshift suppression atother unwanted sites.

For a given system, a selector codon can also include one of the naturalthree base codons, where the endogenous system does not use (or rarelyuses) the natural base codon. For example, this includes a system thatis lacking a tRNA that recognizes the natural three base codon, and/or asystem where the three base codon is a rare codon.

Selector codons optionally include unnatural base pairs. These unnaturalbase pairs further expand the existing genetic alphabet. One extra basepair increases the number of triplet codons from 64 to 125. Propertiesof third base pairs include stable and selective base pairing, efficientenzymatic incorporation into DNA with high fidelity by a polymerase, andthe efficient continued primer extension after synthesis of the nascentunnatural base pair. Descriptions of unnatural base pairs which can beadapted for methods and compositions include, e.g., Hirao, et al.,(2002) An unnatural base pair for incorporating amino acid analoguesinto protein, Nature Biotechnology, 20:177-182. See, also, Wu, Y., etal., (2002) J. Am. Chem. Soc. 124:14626-14630. Other relevantpublications are listed below.

For in vivo usage, the unnatural nucleoside is membrane permeable and isphosphorylated to form the corresponding triphosphate. In addition, theincreased genetic information is stable and not destroyed by cellularenzymes. Previous efforts by Benner and others took advantage ofhydrogen bonding patterns that are different from those in canonicalWatson-Crick pairs, the most noteworthy example of which is theiso-C:iso-G pair. See, e.g., Switzer et al., (1989) J. Am. Chem. Soc.,111:8322; and Piccirilli et al., (1990) Nature, 343:33; Kool, (2000)Curr. Opin. Chem. Biol., 4:602. These bases in general mispair to somedegree with natural bases and cannot be enzymatically replicated. Kooland co-workers demonstrated that hydrophobic packing interactionsbetween bases can replace hydrogen bonding to drive the formation ofbase pair. See, Kool, (2000) Curr. Opin. Chem. Biol., 4:602; and Guckianand Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. In an effort todevelop an unnatural base pair satisfying all the above requirements,Schultz, Romesberg and co-workers have systematically synthesized andstudied a series of unnatural hydrophobic bases. A PICS:PICS self-pairis found to be more stable than natural base pairs, and can beefficiently incorporated into DNA by Klenow fragment of Escherichia coliDNA polymerase I (KF). See, e.g., McMinn et al., (1999) J. Am. Chem.Soc., 121:11585-6; and Ogawa et al., (2000) J. Am. Chem. Soc., 122:3274.A 3MN:3MN self-pair can be synthesized by KF with efficiency andselectivity sufficient for biological function. See, e.g., Ogawa et al.,(2000) J. Am. Chem. Soc., 122:8803. However, both bases act as a chainterminator for further replication. A mutant DNA polymerase has beenrecently evolved that can be used to replicate the PICS self pair. Inaddition, a 7AI self pair can be replicated. See, e.g., Tae et al.,(2001) J. Am. Chem. Soc., 123:7439. A novel metallobase pair, Dipic:Py,has also been developed, which forms a stable pair upon binding Cu(II).See, Meggers et al., (2000) J. Am. Chem. Soc., 122:10714. Becauseextended codons and unnatural codons are intrinsically orthogonal tonatural codons, the methods of the invention can take advantage of thisproperty to generate orthogonal tRNAs for them.

A translational bypassing system can also be used to incorporate anunnatural amino acid in a desired polypeptide. In a translationalbypassing system, a large sequence is incorporated into a gene but isnot translated into protein. The sequence contains a structure thatserves as a cue to induce the ribosome to hop over the sequence andresume translation downstream of the insertion.

In certain embodiments, the protein or polypeptide of interest (orportion thereof) in the methods and/or compositions of the invention isencoded by a nucleic acid. Typically, the nucleic acid comprises atleast one selector codon, at least two selector codons, at least threeselector codons, at least four selector codons, at least five selectorcodons, at least six selector codons, at least seven selector codons, atleast eight selector codons, at least nine selector codons, ten or moreselector codons.

Genes coding for proteins or polypeptides of interest can be mutagenizedusing methods known to one of ordinary skill in the art and describedherein to include, for example, one or more selector codon for theincorporation of an unnatural amino acid. For example, a nucleic acidfor a protein of interest is mutagenized to include one or more selectorcodon, providing for the incorporation of one or more unnatural aminoacids. The invention includes any such variant, including but notlimited to, mutant, versions of any protein, for example, including atleast one unnatural amino acid. Similarly, the invention also includescorresponding nucleic acids, i.e., any nucleic acid with one or moreselector codon that encodes one or more unnatural amino acid.

Nucleic acid molecules encoding a protein of interest such as a FGF-21polypeptide may be readily mutated to introduce a cysteine at anydesired position of the polypeptide. Cysteine is widely used tointroduce reactive molecules, water soluble polymers, proteins, or awide variety of other molecules, onto a protein of interest. Methodssuitable for the incorporation of cysteine into a desired position of apolypeptide are known to those of ordinary skill in the art, such asthose described in U.S. Pat. No. 6,608,183, which is incorporated byreference herein, and standard mutagenesis techniques.

IV. Non-Naturally Encoded Amino Acids

A very wide variety of non-naturally encoded amino acids are suitablefor use in the present invention. Any number of non-naturally encodedamino acids can be introduced into a FGF-21 polypeptide. In general, theintroduced non-naturally encoded amino acids are substantiallychemically inert toward the 20 common, genetically-encoded amino acids(i.e., alanine, arginine, asparagine, aspartic acid, cysteine,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine). In some embodiments, thenon-naturally encoded amino acids include side chain functional groupsthat react efficiently and selectively with functional groups not foundin the 20 common amino acids (including but not limited to, azido,ketone, aldehyde and aminooxy groups) to form stable conjugates. Forexample, a FGF-21 polypeptide that includes a non-naturally encodedamino acid containing an azido functional group can be reacted with apolymer (including but not limited to, poly(ethylene glycol) or,alternatively, a second polypeptide containing an alkyne moiety to forma stable conjugate resulting for the selective reaction of the azide andthe alkyne functional groups to form a Huisgen [3+2] cycloadditionproduct.

The generic structure of an alpha-amino acid is illustrated as follows(Formula I):

A non-naturally encoded amino acid is typically any structure having theabove-listed formula wherein the R group is any substituent other thanone used in the twenty natural amino acids, and may be suitable for usein the present invention. Because the non-naturally encoded amino acidsof the invention typically differ from the natural amino acids only inthe structure of the side chain, the non-naturally encoded amino acidsform amide bonds with other amino acids, including but not limited to,natural or non-naturally encoded, in the same manner in which they areformed in naturally occurring polypeptides. However, the non-naturallyencoded amino acids have side chain groups that distinguish them fromthe natural amino acids. For example, R optionally comprises an alkyl-,aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-,hydrazide, alkenyl, alkynl, ether, thiol, seleno-, sulfonyl-, borate,boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine,aldehyde, ester, thioacid, hydroxylamine, amino group, or the like orany combination thereof. Other non-naturally occurring amino acids ofinterest that may be suitable for use in the present invention include,but are not limited to, amino acids comprising a photoactivatablecross-linker, spin-labeled amino acids, fluorescent amino acids, metalbinding amino acids, metal-containing amino acids, radioactive aminoacids, amino acids with novel functional groups, amino acids thatcovalently or noncovalently interact with other molecules, photocagedand/or photoisomerizable amino acids, amino acids comprising biotin or abiotin analogue, glycosylated amino acids such as a sugar substitutedserine, other carbohydrate modified amino acids, keto-containing aminoacids, amino acids comprising polyethylene glycol or polyether, heavyatom substituted amino acids, chemically cleavable and/or photocleavableamino acids, amino acids with an elongated side chains as compared tonatural amino acids, including but not limited to, polyethers or longchain hydrocarbons, including but not limited to, greater than about 5or greater than about 10 carbons, carbon-linked sugar-containing aminoacids, redox-active amino acids, amino thioacid containing amino acids,and amino acids comprising one or more toxic moiety.

Exemplary non-naturally encoded amino acids that may be suitable for usein the present invention and that are useful for reactions with watersoluble polymers include, but are not limited to, those with carbonyl,aminooxy, hydrazine, hydrazide, semicarbazide, azide and alkyne reactivegroups. In some embodiments, non-naturally encoded amino acids comprisea saccharide moiety. Examples of such amino acids includeN-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine,N-acetyl-L-glucosaminyl-L-threonine,N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L-serine.Examples of such amino acids also include examples where thenaturally-occuring N- or O-linkage between the amino acid and thesaccharide is replaced by a covalent linkage not commonly found innature—including but not limited to, an alkene, an oxime, a thioether,an amide and the like. Examples of such amino acids also includesaccharides that are not commonly found in naturally-occuring proteinssuch as 2-deoxy-glucose, 2-deoxygalactose and the like.

Many of the non-naturally encoded amino acids provided herein arecommercially available, e.g., from Sigma-Aldrich (St. Louis, Mo., USA),Novabiochem (a division of EMD Biosciences, Darmstadt, Germany), orPeptech (Burlington, Mass., USA). Those that are not commerciallyavailable are optionally synthesized as provided herein or usingstandard methods known to those of ordinary skill in the art. Fororganic synthesis techniques, see, e.g., Organic Chemistry by Fessendonand Fessendon, (1982, Second Edition, Willard Grant Press, BostonMass.); Advanced Organic Chemistry by March (Third Edition, 1985, Wileyand Sons, New York); and Advanced Organic Chemistry by Carey andSundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York).See, also, U.S. Pat. Nos. 7,045,337 and 7,083,970, which areincorporated by reference herein. In addition to unnatural amino acidsthat contain novel side chains, unnatural amino acids that may besuitable for use in the present invention also optionally comprisemodified backbone structures, including but not limited to, asillustrated by the structures of Formula II and III:

wherein Z typically comprises OH, NH₂, SH, NH—R′, or S—R′; X and Y,which can be the same or different, typically comprise S or O, and R andR′, which are optionally the same or different, are typically selectedfrom the same list of constituents for the R group described above forthe unnatural amino acids having Formula I as well as hydrogen. Forexample, unnatural amino acids of the invention optionally comprisesubstitutions in the amino or carboxyl group as illustrated by FormulasII and III. Unnatural amino acids of this type include, but are notlimited to, α-hydroxy acids, α-thioacids, α-aminothiocarboxylates,including but not limited to, with side chains corresponding to thecommon twenty natural amino acids or unnatural side chains. In addition,substitutions at the α-carbon optionally include, but are not limitedto, L, D, or α-α-disubstituted amino acids such as D-glutamate,D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and the like. Otherstructural alternatives include cyclic amino acids, such as prolineanalogues as well as 3, 4,6, 7, 8, and 9 membered ring prolineanalogues, β and γ amino acids such as substituted β-alanine and γ-aminobutyric acid.

Many unnatural amino acids are based on natural amino acids, such astyrosine, glutamine, phenylalanine, and the like, and are suitable foruse in the present invention. Tyrosine analogs include, but are notlimited to, para-substituted tyrosines, ortho-substituted tyrosines, andmeta substituted tyrosines, where the substituted tyrosine comprises,including but not limited to, a keto group (including but not limitedto, an acetyl group), a benzoyl group, an amino group, a hydrazine, anhydroxyamine, a thiol group, a carboxy group, an isopropyl group, amethyl group, a C₆-C₂₀ straight chain or branched hydrocarbon, asaturated or unsaturated hydrocarbon, an O-methyl group, a polyethergroup, a nitro group, an alkynyl group or the like. In addition,multiply substituted aryl rings are also contemplated. Glutamine analogsthat may be suitable for use in the present invention include, but arenot limited to, α-hydroxy derivatives, γ-substituted derivatives, cyclicderivatives, and amide substituted glutamine derivatives. Examplephenylalanine analogs that may be suitable for use in the presentinvention include, but are not limited to, para-substitutedphenylalanines, ortho-substituted phenyalanines, and meta-substitutedphenylalanines, where the sub stituent comprises, including but notlimited to, a hydroxy group, a methoxy group, a methyl group, an allylgroup, an aldehyde, an azido, an iodo, a bromo, a keto group (includingbut not limited to, an acetyl group), a benzoyl, an alkynyl group, orthe like. Specific examples of unnatural amino acids that may besuitable for use in the present invention include, but are not limitedto, a p-acetyl-L-phenylalanine, an O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-G1cNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, anisopropyl-L-phenylalanine, and a p-propargyloxy-phenylalanine, and thelike. Examples of structures of a variety of unnatural amino acids thatmay be suitable for use in the present invention are provided in, forexample, WO 2002/085923 entitled “In vivo incorporation of unnaturalamino acids.” See also Kiick et al., (2002) Incorporation of azides intorecombinant proteins for chemoselective modification by the Staudingerligation, PNAS 99:19-24, which is incorporated by reference herein, foradditional methionine analogs. International Application No.PCT/US06/47822 entitled “Compositions Containing, Methods Involving, andUses of Non-natural Amino Acids and Polypeptides,” which is incorporatedby reference herein, describes reductive alkylation of an aromatic aminemoieties, including but not limited to, p-amino-phenylalanine andreductive amination.

In one embodiment, compositions of a FGF-21 polypeptide that include anunnatural amino acid (such as p-(propargyloxy)-phenyalanine) areprovided. Various compositions comprising p-(propargyloxy)-phenyalanineand, including but not limited to, proteins and/or cells, are alsoprovided. In one aspect, a composition that includes thep-(propargyloxy)-phenyalanine unnatural amino acid, further includes anorthogonal tRNA. The unnatural amino acid can be bonded (including butnot limited to, covalently) to the orthogonal tRNA, including but notlimited to, covalently bonded to the orthogonal tRNA though anamino-acyl bond, covalently bonded to a 3′OH or a 2′OH of a terminalribose sugar of the orthogonal tRNA, etc.

The chemical moieties via unnatural amino acids that can be incorporatedinto proteins offer a variety of advantages and manipulations of theprotein. For example, the unique reactivity of a keto functional groupallows selective modification of proteins with any of a number ofhydrazine- or hydroxylamine-containing reagents in vitro and in vivo. Aheavy atom unnatural amino acid, for example, can be useful for phasingX-ray structure data. The site-specific introduction of heavy atomsusing unnatural amino acids also provides selectivity and flexibility inchoosing positions for heavy atoms. Photoreactive unnatural amino acids(including but not limited to, amino acids with benzophenone andarylazides (including but not limited to, phenylazide) side chains), forexample, allow for efficient in vivo and in vitro photocrosslinking ofprotein. Examples of photoreactive unnatural amino acids include, butare not limited to, p-azido-phenylalanine and p-benzoyl-phenylalanine.The protein with the photoreactive unnatural amino acids can then becrosslinked at will by excitation of the photoreactive group-providingtemporal control. In one example, the methyl group of an unnatural aminocan be substituted with an isotopically labeled, including but notlimited to, methyl group, as a probe of local structure and dynamics,including but not limited to, with the use of nuclear magnetic resonanceand vibrational spectroscopy. Alkynyl or azido functional groups, forexample, allow the selective modification of proteins with moleculesthrough a [3+2] cycloaddition reaction.

A non-natural amino acid incorporated into a polypeptide at the aminoterminus can be composed of an R group that is any substituent otherthan one used in the twenty natural amino acids and a 2^(nd) reactivegroup different from the NH₂ group normally present in a-amino acids(see Formula I). A similar non-natural amino acid can be incorporated atthe carboxyl terminus with a 2^(nd) reactive group different from theCOOH group normally present in a-amino acids (see Formula I).

The unnatural amino acids of the invention may be selected or designedto provide additional characteristics unavailable in the twenty naturalamino acids. For example, unnatural amino acid may be optionallydesigned or selected to modify the biological properties of a protein,e.g., into which they are incorporated. For example, the followingproperties may be optionally modified by inclusion of an unnatural aminoacid into a protein: toxicity, biodistribution, solubility, stability,e.g., thermal, hydrolytic, oxidative, resistance to enzymaticdegradation, and the like, facility of purification and processing,structural properties, spectroscopic properties, chemical and/orphotochemical properties, catalytic activity, redox potential,half-life, ability to react with other molecules, e.g., covalently ornoncovalently, and the like.

Structure and Synthesis of Non-Natural Amino Acids: Carbonyl,Carbonyl-Like, Masked Carbonyl, Protected Carbonyl Groups, andHydroxylamine Groups

In some embodiments the present invention provides FGF-21 linked to awater soluble polymer, e.g., a PEG, by an oxime bond.

Many types of non-naturally encoded amino acids are suitable forformation of oxime bonds. These include, but are not limited to,non-naturally encoded amino acids containing a carbonyl, dicarbonyl, orhydroxylamine group. Such amino acids are described in U.S. PatentPublication Nos. 2006/0194256, 2006/0217532, and 2006/0217289 and WO2006/069246 entitled “Compositions containing, methods involving, anduses of non-natural amino acids and polypeptides,” which areincorporated herein by reference in their entirety. Non-naturallyencoded amino acids are also described in U.S. Pat. Nos. 7,083,970 and7,045,337, which are incorporated by reference herein in their entirety.

Some embodiments of the invention utilize FGF-21 polypeptides that aresubstituted at one or more positions with a para-acetylphenylalanineamino acid. The synthesis of p-acetyl-(+/−)-phenylalanine andm-acetyl-(+/−)-phenylalanine are described in Zhang, Z., et al.,Biochemistry 42: 6735-6746 (2003), incorporated by reference. Othercarbonyl- or dicarbonyl-containing amino acids can be similarly preparedby one of ordinary skill in the art. Further, non-limiting examplarysyntheses of non-natural amino acid that are included herein arepresented in FIGS. 4, 24-34 and 36-39 of U.S. Pat. No. 7,083,970, whichis incorporated by reference herein in its entirety.

Amino acids with an electrophilic reactive group allow for a variety ofreactions to link molecules via nucleophilic addition reactions amongothers. Such electrophilic reactive groups include a carbonyl group(including a keto group and a dicarbonyl group), a carbonyl-like group(which has reactivity similar to a carbonyl group (including a ketogroup and a dicarbonyl group) and is structurally similar to a carbonylgroup), a masked carbonyl group (which can be readily converted into acarbonyl group (including a keto group and a dicarbonyl group)), or aprotected carbonyl group (which has reactivity similar to a carbonylgroup (including a keto group and a dicarbonyl group) upondeprotection). Such amino acids include amino acids having the structureof Formula (IV):

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;

J is

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;each R″ is independently H, alkyl, substituted alkyl, or a protectinggroup, or when more than one R″ group is present, two R″ optionally forma heterocycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;each of R₃ and R₄ is independently H, halogen, lower alkyl, orsubstituted lower alkyl, or R₃ and R₄ or two R₃ groups optionally form acycloalkyl or a heterocycloalkyl;or the -A-B-J-R groups together form a bicyclic or tricyclic cycloalkylor heterocycloalkyl comprising at least one carbonyl group, including adicarbonyl group, protected carbonyl group, including a protecteddicarbonyl group, or masked carbonyl group, including a maskeddicarbonyl group;or the -J-R group together forms a monocyclic or bicyclic cycloalkyl orheterocycloalkyl comprising at least one carbonyl group, including adicarbonyl group, protected carbonyl group, including a protecteddicarbonyl group, or masked carbonyl group, including a maskeddicarbonyl group;with a proviso that when A is phenylene and each R₃ is H, B is present;and that when A is —(CH₂)₄— and each R₃ is H, B is not —NHC(O)(CH₂CH₂)—;and that when A and B are absent and each R₃ is H, R is not methyl.

In addition, having the structure of Formula (V) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′) C(O)O—,—S(O)_(k)N(R′)—, —N(R′) C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;with a proviso that when A is phenylene, B is present; and that when Ais —(CH₂)₄—, B is not —NHC(O)(CH₂CH₂)—; and that when A and B areabsent, R is not methyl.

In addition, amino acids having the structure of Formula (VI) areincluded:

wherein:B is a linker selected from the group consisting of lower alkylene,substituted lower alkylene, lower alkenylene, substituted loweralkenylene, lower heteroalkylene, substituted lower heteroalkylene, —O—,—O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or substitutedalkylene)-, —S(O)_(k)— where k is 1, 2, or 3, —S(O)_(k)(alkylene orsubstituted alkylene)-, —C(O)—, —C(O)-(alkylene or substitutedalkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,—NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,—CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,—CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene orsubstituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′) C(S)N(R′)—, —N(R′) S(O)_(k)N(R′)—, —N(R′)—N═,—C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and—C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, orsubstituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;each R_(a) is independently selected from the group consisting of H,halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)kR′ where k is 1, 2, or3, —C(O)N(R′)₂, —OR′, and —S(O)kR′, where each R′ is independently H,alkyl, or substituted alkyl.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected group, carboxylprotected or a salt thereof. In addition, any of the followingnon-natural amino acids may be incorporated into a non-natural aminoacid polypeptide.

In addition, the following amino acids having the structure of Formula(VII) are included:

whereinB is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′) C(O)O—,—S(O)_(k)N(R′)—, —N(R′) C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;each R_(a) is independently selected from the group consisting of H,halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)kR′ where k is 1, 2, or3, —C(O)N(R′)₂, —OR′, and —S(O)kR′, where each R′ is independently H,alkyl, or substituted alkyl; and n is 0 to 8;with a proviso that when A is —(CH₂)₄—, B is not —NHC(O)(CH₂CH₂)—.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition, the following amino acids having the structure of Formula(VIII) are included:

wherein A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′) C(O)O—,—S(O)_(k)N(R′)—, —N(R′) C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide.

In addition, the following amino acids having the structure of Formula(IX) are included:

B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;wherein each R_(a) is independently selected from the group consistingof H, halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)kR′ where k is 1,2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)kR′, where each R′ is independentlyH, alkyl, or substituted alkyl.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition, the following amino acids having the structure of Formula(X) are included:

wherein B is optional, and when present is a linker selected from thegroup consisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′) CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;each R_(a) is independently selected from the group consisting of H,halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)kR′ where k is 1, 2, or3, —C(O)N(R′)₂, —OR′, and —S(O)kR′, where each R′ is independently H,alkyl, or substituted alkyl; and n is 0 to 8.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition to monocarbonyl structures, the non-natural amino acidsdescribed herein may include groups such as dicarbonyl, dicarbonyl like,masked dicarbonyl and protected dicarbonyl groups.

For example, the following amino acids having the structure of Formula(XI) are included:

wherein A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide.

In addition, the following amino acids having the structure of Formula(XII) are included:

B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′) C(O)O—,—S(O)_(k)N(R′)—, —N(R′) C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;wherein each R_(a) is independently selected from the group consistingof H, halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)kR′ where k is 1,2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)kR′, where each R′ is independentlyH, alkyl, or substituted alkyl.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition, the following amino acids having the structure of Formula(XIII) are included:

wherein B is optional, and when present is a linker selected from thegroup consisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′) CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;each R_(a) is independently selected from the group consisting of H,halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)kR′ where k is 1, 2, or3, —C(O)N(R′)₂, —OR′, and —S(O)kR′, where each R′ is independently H,alkyl, or substituted alkyl; and n is 0 to 8.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition, the following amino acids having the structure of Formula(XIV) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;X₁ is C, S, or S(O); and L is alkylene, substituted alkylene,N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H, alkyl,substituted alkyl, cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XIV-A) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;L is alkylene, substituted alkylene, N(R′)(alkylene) orN(R′)(substituted alkylene), where R′ is H, alkyl, substituted alkyl,cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XIV-B) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;L is alkylene, substituted alkylene, N(R′)(alkylene) orN(R′)(substituted alkylene), where R′ is H, alkyl, substituted alkyl,cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XV) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;X₁ is C, S, or S(O); and n is 0, 1, 2, 3, 4, or 5; and each R⁸ and R⁹ oneach CR⁸R⁹ group is independently selected from the group consisting ofH, alkoxy, alkylamine, halogen, alkyl, aryl, or any R⁸ and R⁹ cantogether form ═O or a cycloalkyl, or any to adjacent R⁸ groups cantogether form a cycloalkyl.

In addition, the following amino acids having the structure of Formula(XV-A) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;n is 0, 1, 2, 3, 4, or 5; and each R⁸ and R⁹ on each CR⁸R⁹ group isindependently selected from the group consisting of H, alkoxy,alkylamine, halogen, alkyl, aryl, or any R⁸ and R⁹ can together form ═Oor a cycloalkyl, or any to adjacent R⁸ groups can together form acycloalkyl.

In addition, the following amino acids having the structure of Formula(XV-B) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;n is 0, 1, 2, 3, 4, or 5; and each R⁸ and R⁹ on each CR⁸R⁹ group isindependently selected from the group consisting of H, alkoxy,alkylamine, halogen, alkyl, aryl, or any R⁸ and R⁹ can together form ═Oor a cycloalkyl, or any to adjacent R⁸ groups can together form acycloalkyl.

In addition, the following amino acids having the structure of Formula(XVI) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;X₁ is C, S, or S(O); and L is alkylene, substituted alkylene,N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H, alkyl,substituted alkyl, cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XVI-A) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;L is alkylene, substituted alkylene, N(R′)(alkylene) orN(R′)(substituted alkylene), where R′ is H, alkyl, substituted alkyl,cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XVI-B) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;L is alkylene, substituted alkylene, N(R′)(alkylene) orN(R′)(substituted alkylene), where R′ is H, alkyl, substituted alkyl,cycloalkyl, or substituted cycloalkyl.

In addition, amino acids having the structure of Formula (XVII) areincluded:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;

M is —C(R₃)—,

where (a) indicates bonding to the A group and (b) indicates bonding torespective carbonyl groups, R₃ and R₄ are independently chosen from H,halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl, or R₃ and R₄ or two R₃ groups or two R₄ groups optionallyform a cycloalkyl or a heterocycloalkyl;R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl;T₃ is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl, substitutedalkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide.

In addition, amino acids having the structure of Formula (XVIII) areincluded:

wherein:

M is —C(R₃)—,

where (a) indicates bonding to the A group and (b) indicates bonding torespective carbonyl groups, R₃ and R₄ are independently chosen from H,halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl, or R₃ and R₄ or two R₃ groups or two R₄ groups optionallyform a cycloalkyl or a heterocycloalkyl;R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl;T₃ is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl, substitutedalkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;each R_(a) is independently selected from the group consisting of H,halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2,or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is independentlyH, alkyl, or substituted alkyl.

In addition, amino acids having the structure of Formula (XIX) areincluded:

wherein:R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl; and

T₃ is O, or S.

In addition, amino acids having the structure of Formula (XX) areincluded:

wherein:R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl.

In addition, the following amino acids having structures of Formula(XXI) are included:

In some embodiments, a polypeptide comprising a non-natural amino acidis chemically modified to generate a reactive carbonyl or dicarbonylfunctional group. For instance, an aldehyde functionality useful forconjugation reactions can be generated from a functionality havingadjacent amino and hydroxyl groups. Where the biologically activemolecule is a polypeptide, for example, an N-terminal serine orthreonine (which may be normally present or may be exposed via chemicalor enzymatic digestion) can be used to generate an aldehydefunctionality under mild oxidative cleavage conditions using periodate.See, e.g., Gaertner, et. al., Bioconjug. Chem. 3: 262-268 (1992);Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3:138-146 (1992); Gaertneret al., J. Biol. Chem. 269:7224-7230 (1994). However, methods known inthe art are restricted to the amino acid at the N-terminus of thepeptide or protein.

In the present invention, a non-natural amino acid bearing adjacenthydroxyl and amino groups can be incorporated into the polypeptide as a“masked” aldehyde functionality. For example, 5-hydroxylysine bears ahydroxyl group adjacent to the epsilon amine. Reaction conditions forgenerating the aldehyde typically involve addition of molar excess ofsodium metaperiodate under mild conditions to avoid oxidation at othersites within the polypeptide. The pH of the oxidation reaction istypically about 7.0. A typical reaction involves the addition of about1.5 molar excess of sodium meta periodate to a buffered solution of thepolypeptide, followed by incubation for about 10 minutes in the dark.See, e.g. U.S. Pat. No. 6,423,685.

The carbonyl or dicarbonyl functionality can be reacted selectively witha hydroxylamine-containing reagent under mild conditions in aqueoussolution to form the corresponding oxime linkage that is stable underphysiological conditions. See, e.g., Jencks, W. P., J. Am. Chem. Soc.81, 475-481 (1959); Shao, J. and Tam, J. P., J. Am. Chem. Soc.117:3893-3899 (1995). Moreover, the unique reactivity of the carbonyl ordicarbonyl group allows for selective modification in the presence ofthe other amino acid side chains. See, e.g., Cornish, V. W., et al., J.Am. Chem. Soc. 118:8150-8151 (1996); Geoghegan, K. F. & Stroh, J. G.,Bioconjug. Chem. 3:138-146 (1992); Mahal, L. K., et al., Science276:1125-1128 (1997).

Structure and Synthesis of Non-Natural Amino Acids:Hydroxylamine-Containing Amino Acids

U.S. Provisional Patent Application No. 60/638,418 is incorporated byreference in its entirety. Thus, the disclosures provided in Section V(entitled “Non-natural Amino Acids”), Part B (entitled “Structure andSynthesis of Non-Natural Amino Acids: Hydroxylamine-Containing AminoAcids”), in U.S. Provisional Patent Application No. 60/638,418 applyfully to the methods, compositions (including Formulas I-XXXV),techniques and strategies for making, purifying, characterizing, andusing non-natural amino acids, non-natural amino acid polypeptides andmodified non-natural amino acid polypeptides described herein to thesame extent as if such disclosures were fully presented herein. U.S.Patent Publication Nos. 2006/0194256, 2006/0217532, and 2006/0217289 andWO 2006/069246 entitled “Compositions containing, methods involving, anduses of non-natural amino acids and polypeptides,” are also incorporatedherein by reference in their entirety.

Chemical Synthesis of Unnatural Amino Acids

Many of the unnatural amino acids suitable for use in the presentinvention are commercially available, e.g., from Sigma (USA) or Aldrich(Milwaukee, Wis., USA). Those that are not commercially available areoptionally synthesized as provided herein or as provided in variouspublications or using standard methods known to those of ordinary skillin the art. For organic synthesis techniques, see, e.g., OrganicChemistry by Fessendon and Fessendon, (1982, Second Edition, WillardGrant Press, Boston Mass.); Advanced Organic Chemistry by March (ThirdEdition, 1985, Wiley and Sons, New York); and Advanced Organic Chemistryby Carey and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press,New York). Additional publications describing the synthesis of unnaturalamino acids include, e.g., WO 2002/085923 entitled “In vivoincorporation of Unnatural Amino Acids;” Matsoukas et al., (1995) J.Med. Chem., 38, 4660-4669; King, F.E. & Kidd, D.A.A. (1949) A NewSynthesis of Glutamine and of γ-Dipeptides of Glutamic Acid fromPhthylated Intermediates. J. Chem. Soc., 3315-3319; Friedman, O.M. &Chatterrji, R. (1959) Synthesis of Derivatives of Glutamine as ModelSubstrates for Anti-Tumor Agents. J. Am. Chem. Soc. 81, 3750-3752;Craig, J. C. et al. (1988) Absolute Configuration of the Enantiomers of7-Chloro-4 [[4-(diethylamino)-1-methylbutyl]amino]quinoline(Chloroquine). J. Org. Chem. 53, 1167-1170; Azoulay, M., Vilmont, M. &Frappier, F. (1991) Glutamine analogues as Potential Antimalarials, Eur.J. Med. Chem. 26, 201-5; Koskinen, A.M.P. & Rapoport, H. (1989)Synthesis of 4-Substituted Prolines as Conformationally ConstrainedAmino Acid Analogues. J. Org. Chem. 54, 1859-1866; Christie, B.D. &Rapoport, H. (1985) Synthesis of Optically Pure Pipecolates fromL-Asparagine. Application to the Total Synthesis of (+)-Apovincaminethrough Amino Acid Decarbonylation and Iminium Ion Cyclization. J. Org.Chem. 50:1239-1246; Barton et al., (1987) Synthesis of Novelalpha-Amino-Acids and Derivatives Using Radical Chemistry: Synthesis ofL- and D-alpha-Amino-Adipic Acids, L-alpha-aminopimelic Acid andAppropriate Unsaturated Derivatives. Tetrahedron 43:4297-4308; and,Subasinghe et al., (1992) Quisqualic acid analogues: synthesis ofbeta-heterocyclic 2-aminopropanoic acid derivatives and their activityat a novel quisqualate-sensitized site. J. Med. Chem. 35:4602-7. Seealso, U.S. Patent Publication No. US 2004/0198637 entitled “ProteinArrays,” which is incorporated by reference herein.

A. Carbonyl reactive groups

Amino acids with a carbonyl reactive group allow for a variety ofreactions to link molecules (including but not limited to, PEG or otherwater soluble molecules) via nucleophilic addition or aldol condensationreactions among others.

Exemplary carbonyl-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; R₂ is H, alkyl, aryl, substituted alkyl, andsubstituted aryl; and R₃ is H, an amino acid, a polypeptide, or an aminoterminus modification group, and R₄ is H, an amino acid, a polypeptide,or a carboxy terminus modification group. In some embodiments, n is 1,R₁ is phenyl and R₂ is a simple alkyl (i.e., methyl, ethyl, or propyl)and the ketone moiety is positioned in the para position relative to thealkyl side chain. In some embodiments, n is 1, R₁ is phenyl and R₂ is asimple alkyl (i.e., methyl, ethyl, or propyl) and the ketone moiety ispositioned in the meta position relative to the alkyl side chain.

The synthesis of p-acetyl-(+/−)-phenylalanine andm-acetyl-(+/−)-phenylalanine is described in Zhang, Z., et al.,Biochemistry 42: 6735-6746 (2003), which is incorporated by referenceherein. Other carbonyl-containing amino acids can be similarly preparedby one of ordinary skill in the art.

In some embodiments, a polypeptide comprising a non-naturally encodedamino acid is chemically modified to generate a reactive carbonylfunctional group. For instance, an aldehyde functionality useful forconjugation reactions can be generated from a functionality havingadjacent amino and hydroxyl groups. Where the biologically activemolecule is a polypeptide, for example, an N-terminal serine orthreonine (which may be normally present or may be exposed via chemicalor enzymatic digestion) can be used to generate an aldehydefunctionality under mild oxidative cleavage conditions using periodate.See, e.g., Gaertner, et al., Bioconjug. Chem. 3: 262-268 (1992);Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3:138-146 (1992); Gaertneret al., I Biol. Chem. 269:7224-7230 (1994). However, methods known inthe art are restricted to the amino acid at the N-terminus of thepeptide or protein.

In the present invention, a non-naturally encoded amino acid bearingadjacent hydroxyl and amino groups can be incorporated into thepolypeptide as a “masked” aldehyde functionality. For example,5-hydroxylysine bears a hydroxyl group adjacent to the epsilon amine.Reaction conditions for generating the aldehyde typically involveaddition of molar excess of sodium metaperiodate under mild conditionsto avoid oxidation at other sites within the polypeptide. The pH of theoxidation reaction is typically about 7.0. A typical reaction involvesthe addition of about 1.5 molar excess of sodium meta periodate to abuffered solution of the polypeptide, followed by incubation for about10 minutes in the dark. See, e.g. U.S. Pat. No. 6,423,685, which isincorporated by reference herein.

The carbonyl functionality can be reacted selectively with a hydrazine-,hydrazide-, hydroxylamine-, or semicarbazide-containing reagent undermild conditions in aqueous solution to form the corresponding hydrazone,oxime, or semicarbazone linkages, respectively, that are stable underphysiological conditions. See, e.g., Jencks, W. P., J. Am. Chem. Soc.81, 475-481 (1959); Shao, J. and Tam, J. P., J. Am. Chem. Soc.117:3893-3899 (1995). Moreover, the unique reactivity of the carbonylgroup allows for selective modification in the presence of the otheramino acid side chains. See, e.g., Cornish, V. W., et al., J. Am. Chem.Soc. 118:8150-8151 (1996); Geoghegan, K. F. & Stroh, J. G., Bioconjug.Chem. 3:138-146 (1992); Mahal, L. K., et al., Science 276:1125-1128(1997).

B. Hydrazine, hydrazide or semicarbazide reactive groups

Non-naturally encoded amino acids containing a nucleophilic group, suchas a hydrazine, hydrazide or semicarbazide, allow for reaction with avariety of electrophilic groups to form conjugates (including but notlimited to, with PEG or other water soluble polymers).

Exemplary hydrazine, hydrazide or semicarbazide-containing amino acidscan be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X, is O, N, or S or not present; R₂ isH, an amino acid, a polypeptide, or an amino terminus modificationgroup, and R₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, n is 4, R₁ is not present, and X is N. In someembodiments, n is 2, R₁ is not present, and X is not present. In someembodiments, n is 1, R₁ is phenyl, X is O, and the oxygen atom ispositioned para to the alphatic group on the aryl ring.

Hydrazide-, hydrazine-, and semicarbazide-containing amino acids areavailable from commercial sources. For instance, L-glutamate-y-hydrazideis available from Sigma Chemical (St. Louis, Mo.). Other amino acids notavailable commercially can be prepared by one of ordinary skill in theart. See, e.g., U.S. Pat. No. 6,281,211, which is incorporated byreference herein.

Polypeptides containing non-naturally encoded amino acids that bearhydrazide, hydrazine or semicarbazide functionalities can be reactedefficiently and selectively with a variety of molecules that containaldehydes or other functional groups with similar chemical reactivity.See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc. 117:3893-3899 (1995).The unique reactivity of hydrazide, hydrazine and semicarbazidefunctional groups makes them significantly more reactive towardaldehydes, ketones and other electrophilic groups as compared to thenucleophilic groups present on the 20 common amino acids (including butnot limited to, the hydroxyl group of serine or threonine or the aminogroups of lysine and the N-terminus).

C. Aminooxy-containing amino acids

Non-naturally encoded amino acids containing an aminooxy (also called ahydroxylamine) group allow for reaction with a variety of electrophilicgroups to form conjugates (including but not limited to, with PEG orother water soluble polymers). Like hydrazines, hydrazides andsemicarbazides, the enhanced nucleophilicity of the aminooxy grouppermits it to react efficiently and selectively with a variety ofmolecules that contain aldehydes or other functional groups with similarchemical reactivity. See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc.117:3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34:727-736 (2001). Whereas the result of reaction with a hydrazine group isthe corresponding hydrazone, however, an oxime results generally fromthe reaction of an aminooxy group with a carbonyl-containing group suchas a ketone.

Exemplary amino acids containing aminooxy groups can be represented asfollows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X is O, N, S or not present; m is 0-10;Y=C(O) or not present; R₂ is H, an amino acid, a polypeptide, or anamino terminus modification group, and R₃ is H, an amino acid, apolypeptide, or a carboxy terminus modification group. In someembodiments, n is 1, R₁ is phenyl, X is O, m is 1, and Y is present. Insome embodiments, n is 2, R₁ and X are not present, m is 0, and Y is notpresent.

Aminooxy-containing amino acids can be prepared from readily availableamino acid precursors (homoserine, serine and threonine). See, e.g., M.Carrasco and R. Brown, J. Org. Chem. 68: 8853-8858 (2003). Certainaminooxy-containing amino acids, such as L-2-amino-4-(aminooxy)butyricacid), have been isolated from natural sources (Rosenthal, G., Life Sci.60: 1635-1641 (1997). Other aminooxy-containing amino acids can beprepared by one of ordinary skill in the art.

D. Azide and alkyne reactive groups

The unique reactivity of azide and alkyne functional groups makes themextremely useful for the selective modification of polypeptides andother biological molecules. Organic azides, particularly alphaticazides, and alkynes are generally stable toward common reactive chemicalconditions. In particular, both the azide and the alkyne functionalgroups are inert toward the side chains (i.e., R groups) of the 20common amino acids found in naturally-occuring polypeptides. Whenbrought into close proximity, however, the “spring-loaded” nature of theazide and alkyne groups is revealed and they react selectively andefficiently via Huisgen [3+2] cycloaddition reaction to generate thecorresponding triazole. See, e.g., Chin J., et al., Science 301:964-7(2003); Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Chin,J.W., et al., J. Am. Chem. Soc. 124:9026-9027 (2002).

Because the Huisgen cycloaddition reaction involves a selectivecycloaddition reaction (see, e.g., Padwa, A., in COMPREHENSIVE ORGANICSYNTHESIS, Vol. 4, (ed. Trost, B. M., 1991), p. 1069-1109; Huisgen, R.in 1,3-DIPOLAR CYCLOADDITION CHEMISTRY, (ed. Padwa, A., 1984), p. 1-176)rather than a nucleophilic substitution, the incorporation ofnon-naturally encoded amino acids bearing azide and alkyne-containingside chains permits the resultant polypeptides to be modifiedselectively at the position of the non-naturally encoded amino acid.Cycloaddition reaction involving azide or alkyne-containing FGF-21polypeptide can be carried out at room temperature under aqueousconditions by the addition of Cu(II) (including but not limited to, inthe form of a catalytic amount of CuSO₄) in the presence of a reducingagent for reducing Cu(II) to Cu(I), in situ, in catalytic amount. See,e.g., Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Tornoe,C. W., et al., J. Org. Chem. 67:3057-3064 (2002); Rostovtsev, et al.,Angew. Chem. Int. Ed. 41:2596-2599 (2002). Exemplary reducing agentsinclude, including but not limited to, ascorbate, metallic copper,quinine, hydroquinone, vitamin K, glutathione, cysteine, Fe²⁺, Co²⁺, andan applied electric potential.

In some cases, where a Huisgen [3+2] cycloaddition reaction between anazide and an alkyne is desired, the FGF-21 polypeptide comprises anon-naturally encoded amino acid comprising an alkyne moiety and thewater soluble polymer to be attached to the amino acid comprises anazide moiety. Alternatively, the converse reaction (i.e., with the azidemoiety on the amino acid and the alkyne moiety present on the watersoluble polymer) can also be performed.

The azide functional group can also be reacted selectively with a watersoluble polymer containing an aryl ester and appropriatelyfunctionalized with an aryl phosphine moiety to generate an amidelinkage. The aryl phosphine group reduces the azide in situ and theresulting amine then reacts efficiently with a proximal ester linkage togenerate the corresponding amide. See, e.g., E. Saxon and C. Bertozzi,Science 287, 2007-2010 (2000). The azide-containing amino acid can beeither an alkyl azide (including but not limited to,2-amino-6-azido-1-hexanoic acid) or an aryl azide(p-azido-phenylalanine).

Exemplary water soluble polymers containing an aryl ester and aphosphine moiety can be represented as follows:

wherein X can be O, N, S or not present, Ph is phenyl, W is a watersoluble polymer and R can be H, alkyl, aryl, substituted alkyl andsubstituted aryl groups. Exemplary R groups include but are not limitedto —CH₂, —C(CH₃) 3, —OR′, —NR′R″, —SR′, -halogen, —C(O)R′, —CONR′R″,—S(O)₂R′, —S(O)₂NR′R″, —CN and —NO₂. R′, R″, R′″ and R″″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R″″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF₃ and —CH₂CF₃) and acyl (including but not limited to,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

The azide functional group can also be reacted selectively with a watersoluble polymer containing a thioester and appropriately functionalizedwith an aryl phosphine moiety to generate an amide linkage. The arylphosphine group reduces the azide in situ and the resulting amine thenreacts efficiently with the thioester linkage to generate thecorresponding amide. Exemplary water soluble polymers containing athioester and a phosphine moiety can be represented as follows:

wherein n is 1-10; X can be O, N, S or not present, Ph is phenyl, and Wis a water soluble polymer.

Exemplary alkyne-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X is O, N, S or not present; m is 0-10,R₂ is H, an amino acid, a polypeptide, or an amino terminus modificationgroup, and R₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group. In some embodiments, n is 1, R₁ is phenyl, X is notpresent, m is 0 and the acetylene moiety is positioned in the paraposition relative to the alkyl side chain. In some embodiments, n is 1,R₁ is phenyl, X is O, m is 1 and the propargyloxy group is positioned inthe para position relative to the alkyl side chain (i.e.,0-propargyl-tyrosine). In some embodiments, n is 1, R₁ and X are notpresent and m is 0 (i.e., proparylglycine).

Alkyne-containing amino acids are commercially available. For example,propargylglycine is commercially available from Peptech (Burlington,Mass.). Alternatively, alkyne-containing amino acids can be preparedaccording to standard methods. For instance, p-propargyloxyphenylalaninecan be synthesized, for example, as described in Deiters, A., et al., J.Am. Chem. Soc. 125: 11782-11783 (2003), and 4-alkynyl-L-phenylalaninecan be synthesized as described in Kayser, B., et al., Tetrahedron53(7): 2475-2484 (1997). Other alkyne-containing amino acids can beprepared by one of ordinary skill in the art.

Exemplary azide-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, substitutedaryl or not present; X is O, N, S or not present; m is 0-10; R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group. In some embodiments, n is 1, R₁ is phenyl, X is notpresent, m is 0 and the azide moiety is positioned para to the alkylside chain. In some embodiments, n is 0-4 and R₁ and X are not present,and m=0. In some embodiments, n is 1, R₁ is phenyl, X is O, m is 2 andthe β-azidoethoxy moiety is positioned in the para position relative tothe alkyl side chain.

Azide-containing amino acids are available from commercial sources. Forinstance, 4-azidophenylalanine can be obtained from Chem-ImpexInternational, Inc. (Wood Dale, Ill.). For those azide-containing aminoacids that are not commercially available, the azide group can beprepared relatively readily using standard methods known to those ofordinary skill in the art, including but not limited to, viadisplacement of a suitable leaving group (including but not limited to,halide, mesylate, tosylate) or via opening of a suitably protectedlactone. See, e.g., Advanced Organic Chemistry by March (Third Edition,1985, Wiley and Sons, New York).

E. Aminothiol reactive groups

The unique reactivity of beta-substituted aminothiol functional groupsmakes them extremely useful for the selective modification ofpolypeptides and other biological molecules that contain aldehyde groupsvia formation of the thiazolidine. See, e.g., J. Shao and J. Tam, J. Am.Chem. Soc. 1995, 117 (14) 3893-3899. In some embodiments,beta-substituted aminothiol amino acids can be incorporated into FGF-21polypeptides and then reacted with water soluble polymers comprising analdehyde functionality. In some embodiments, a water soluble polymer,drug conjugate or other payload can be coupled to a FGF-21 polypeptidecomprising a beta-substituted aminothiol amino acid via formation of thethiazolidine.

F. Additional reactive groups

Additional reactive groups and non-naturally encoded amino acids thatcan be incorporated into FGF-21 polypeptides of the invention aredescribed in the following patent applications which are allincorporated by reference in their entirety herein: U.S. PatentPublication No. 2006/0194256, U.S. Patent Publication No. 2006/0217532,U.S. Patent Publication No. 2006/0217289, U.S. Provisional Patent No.60/755,338; U.S. Provisional Patent No. 60/755,711; U.S. ProvisionalPatent No. 60/755,018; International Patent Application No.PCT/US06/49397; WO 2006/069246; U.S. Provisional Patent No. 60/743,041;U.S. Provisional Patent No. 60/743,040; International Patent ApplicationNo. PCT/US06/47822; U.S. Provisional Patent No. 60/882,819; U.S.Provisional Patent No. 60/882,500; and U.S. Provisional Patent No.60/870,594.

Cellular Uptake of Unnatural Amino Acids

Unnatural amino acid uptake by a cell is one issue that is typicallyconsidered when designing and selecting unnatural amino acids, includingbut not limited to, for incorporation into a protein. For example, thehigh charge density of a-amino acids suggests that these compounds areunlikely to be cell permeable. Natural amino acids are taken up into theeukaryotic cell via a collection of protein-based transport systems. Arapid screen can be done which assesses which unnatural amino acids, ifany, are taken up by cells. See, e.g., the toxicity assays in, e.g.,U.S. Patent Publication No. US 2004/0198637 entitled “Protein Arrays”which is incorporated by reference herein; and Liu, D.R. & Schultz, P.G.(1999) Progress toward the evolution of an organism with an expandedgenetic code. PNAS United States 96:4780-4785. Although uptake is easilyanalyzed with various assays, an alternative to designing unnaturalamino acids that are amenable to cellular uptake pathways is to providebiosynthetic pathways to create amino acids in vivo.

Biosynthesis of Unnatural Amino Acids

Many biosynthetic pathways already exist in cells for the production ofamino acids and other compounds. While a biosynthetic method for aparticular unnatural amino acid may not exist in nature, including butnot limited to, in a cell, the invention provides such methods. Forexample, biosynthetic pathways for unnatural amino acids are optionallygenerated in host cell by adding new enzymes or modifying existing hostcell pathways. Additional new enzymes are optionally naturally occurringenzymes or artificially evolved enzymes. For example, the biosynthesisof p-aminophenylalanine (as presented in an example in WO 2002/085923entitled “In vivo incorporation of unnatural amino acids”) relies on theaddition of a combination of known enzymes from other organisms. Thegenes for these enzymes can be introduced into a eukaryotic cell bytransforming the cell with a plasmid comprising the genes. The genes,when expressed in the cell, provide an enzymatic pathway to synthesizethe desired compound. Examples of the types of enzymes that areoptionally added are provided in the examples below. Additional enzymessequences are found, for example, in Genbank. Artificially evolvedenzymes are also optionally added into a cell in the same manner. Inthis manner, the cellular machinery and resources of a cell aremanipulated to produce unnatural amino acids.

A variety of methods are available for producing novel enzymes for usein biosynthetic pathways or for evolution of existing pathways. Forexample, recursive recombination, including but not limited to, asdeveloped by Maxygen, Inc. (available on the World Wide Web atmaxygen.com), is optionally used to develop novel enzymes and pathways.See, e.g., Stemmer (1994), Rapid evolution of a protein in vitro by DNAshuffling, Nature 370(4):389-391; and, Stemmer, (1994), DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution, Proc. Natl. Acad. Sci. USA., 91:10747-10751.Similarly DesignPath™ developed by Genencor (available on the World WideWeb at genencor.com) is optionally used for metabolic pathwayengineering, including but not limited to, to engineer a pathway tocreate O-methyl-L-tyrosine in a cell. This technology reconstructsexisting pathways in host organisms using a combination of new genes,including but not limited to, those identified through functionalgenomics, and molecular evolution and design. Diversa Corporation(available on the World Wide Web at diversa.com) also providestechnology for rapidly screening libraries of genes and gene pathways,including but not limited to, to create new pathways.

Typically, the unnatural amino acid produced with an engineeredbiosynthetic pathway of the invention is produced in a concentrationsufficient for efficient protein biosynthesis, including but not limitedto, a natural cellular amount, but not to such a degree as to affect theconcentration of the other amino acids or exhaust cellular resources.Typical concentrations produced in vivo in this manner are about 10 mMto about 0.05 mM. Once a cell is transformed with a plasmid comprisingthe genes used to produce enzymes desired for a specific pathway and anunnatural amino acid is generated, in vivo selections are optionallyused to further optimize the production of the unnatural amino acid forboth ribosomal protein synthesis and cell growth.

Polypeptides with Unnatural Amino Acids

The incorporation of an unnatural amino acid can be done for a varietyof purposes, including but not limited to, tailoring changes in proteinstructure and/or function, changing size, acidity, nucleophilicity,hydrogen bonding, hydrophobicity, accessibility of protease targetsites, targeting to a moiety (including but not limited to, for aprotein array), adding a biologically active molecule, attaching apolymer, attaching a radionuclide, modulating serum half-life,modulating tissue penetration (e.g. tumors), modulating activetransport, modulating tissue, cell or organ specificity or distribution,modulating immunogenicity, modulating protease resistance, etc. Proteinsthat include an unnatural amino acid can have enhanced or even entirelynew catalytic or biophysical properties. For example, the followingproperties are optionally modified by inclusion of an unnatural aminoacid into a protein: toxicity, biodistribution, structural properties,spectroscopic properties, chemical and/or photochemical properties,catalytic ability, half-life (including but not limited to, serumhalf-life), ability to react with other molecules, including but notlimited to, covalently or noncovalently, and the like. The compositionsincluding proteins that include at least one unnatural amino acid areuseful for, including but not limited to, novel therapeutics,diagnostics, catalytic enzymes, industrial enzymes, binding proteins(including but not limited to, antibodies), and including but notlimited to, the study of protein structure and function. See, e.g.,Dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structureand Function, Current Opinion in Chemical Biology, 4:645-652.

In one aspect of the invention, a composition includes at least oneprotein with at least one, including but not limited to, at least two,at least three, at least four, at least five, at least six, at leastseven, at least eight, at least nine, or at least ten or more unnaturalamino acids. The unnatural amino acids can be the same or different,including but not limited to, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 or more different sites in the protein that comprise 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 or more different unnatural amino acids. In anotheraspect, a composition includes a protein with at least one, but fewerthan all, of a particular amino acid present in the protein issubstituted with the unnatural amino acid. For a given protein with morethan one unnatural amino acids, the unnatural amino acids can beidentical or different (including but not limited to, the protein caninclude two or more different types of unnatural amino acids, or caninclude two of the same unnatural amino acid). For a given protein withmore than two unnatural amino acids, the unnatural amino acids can bethe same, different or a combination of a multiple unnatural amino acidof the same kind with at least one different unnatural amino acid.

Proteins or polypeptides of interest with at least one unnatural aminoacid are a feature of the invention. The invention also includespolypeptides or proteins with at least one unnatural amino acid producedusing the compositions and methods of the invention. An excipient(including but not limited to, a pharmaceutically acceptable excipient)can also be present with the protein.

By producing proteins or polypeptides of interest with at least oneunnatural amino acid in eukaryotic cells, proteins or polypeptides willtypically include eukaryotic post-translational modifications. Incertain embodiments, a protein includes at least one unnatural aminoacid and at least one post-translational modification that is made invivo by a eukaryotic cell, where the post-translational modification isnot made by a prokaryotic cell. For example, the post-translationmodification includes, including but not limited to, acetylation,acylation, lipid-modification, palmitoylation, palmitate addition,phosphorylation, glycolipid-linkage modification, glycosylation, and thelike. In one aspect, the post-translational modification includesattachment of an oligosaccharide (including but not limited to,(GlcNAc-Man)₂-Man-GlcNAc-GlcNAc)) to an asparagine by aGlcNAc-asparagine linkage. See Table 1 which lists some examples ofN-linked oligosaccharides of eukaryotic proteins (additional residuescan also be present, which are not shown). In another aspect, thepost-translational modification includes attachment of anoligosaccharide (including but not limited to, Gal-GalNAc, Gal-GlcNAc,etc.) to a serine or threonine by a GalNAc-serine or GalNAc-threoninelinkage, or a GlcNAc-serine or a GlcNAc-threonine linkage.

TABLE 1 EXAMPLES OF OLIGOSACCHARIDES THROUGH GlcNAc-LINKAGE Type BaseStructure High- man- nose

Hybrid

Com- plex

Xylose

In yet another aspect, the post-translation modification includesproteolytic processing of precursors (including but not limited to,calcitonin precursor, calcitonin gene-related peptide precursor,preproparathyroid hormone, preproinsulin, proinsulin,prepro-opiomelanocortin, pro-opiomelanocortin and the like), assemblyinto a multisubunit protein or macromolecular assembly, translation toanother site in the cell (including but not limited to, to organelles,such as the endoplasmic reticulum, the Golgi apparatus, the nucleus,lysosomes, peroxisomes, mitochondria, chloroplasts, vacuoles, etc., orthrough the secretory pathway). In certain embodiments, the proteincomprises a secretion or localization sequence, an epitope tag, a FLAGtag, a polyhistidine tag, a GST fusion, or the like.

One advantage of an unnatural amino acid is that it presents additionalchemical moieties that can be used to add additional molecules. Thesemodifications can be made in vivo in a eukaryotic or non-eukaryoticcell, or in vitro. Thus, in certain embodiments, the post-translationalmodification is through the unnatural amino acid. For example, thepost-translational modification can be through anucleophilic-electrophilic reaction. Most reactions currently used forthe selective modification of proteins involve covalent bond formationbetween nucleophilic and electrophilic reaction partners, including butnot limited to the reaction of a-haloketones with histidine or cysteineside chains. Selectivity in these cases is determined by the number andaccessibility of the nucleophilic residues in the protein. In proteinsof the invention, other more selective reactions can be used such as thereaction of an unnatural keto-amino acid with hydrazides or aminooxycompounds, in vitro and in vivo. See, e.g., Cornish, et al., (1996) J.Am. Chem. Soc., 118:8150-8151; Mahal, et al., (1997) Science,276:1125-1128; Wang, et al., (2001) Science 292:498-500; Chin, et al.,(2002) J. Am. Chem. Soc. 124:9026-9027; Chin, et al., (2002) Proc. Natl.Acad. Sci., 99:11020-11024; Wang, et al., (2003) Proc. Natl. Acad. Sci.,100:56-61; Zhang, et al., (2003) Biochemistry, 42:6735-6746; and, Chin,et al., (2003) Science, 301:964-7, all of which are incorporated byreference herein. This allows the selective labeling of virtually anyprotein with a host of reagents including fluorophores, crosslinkingagents, saccharide derivatives and cytotoxic molecules. See also, U.S.Pat. No. 6,927,042 entitled “Glycoprotein synthesis,” which isincorporated by reference herein. Post-translational modifications,including but not limited to, through an azido amino acid, can also madethrough the Staudinger ligation (including but not limited to, withtriarylphosphine reagents). See, e.g., Kiick et al., (2002)Incorporation of azides into recombinant proteins for chemoselectivemodification by the Staudinger ligation, PNAS 99:19-24.

This invention provides another highly efficient method for theselective modification of proteins, which involves the geneticincorporation of unnatural amino acids, including but not limited to,containing an azide or alkynyl moiety into proteins in response to aselector codon. These amino acid side chains can then be modified by,including but not limited to, a Huisgen [3+2] cycloaddition reaction(see, e.g., Padwa, A. in Comprehensive Organic Synthesis, Vol. 4, (1991)Ed. Trost, B. M., Pergamon, Oxford, p. 1069-1109; and, Huisgen, R. in1,3-Dipolar Cycloaddition Chemistry, (1984) Ed. Padwa, A., Wiley, NewYork, p. 1-176) with, including but not limited to, alkynyl or azidederivatives, respectively. Because this method involves a cycloadditionrather than a nucleophilic substitution, proteins can be modified withextremely high selectivity. This reaction can be carried out at roomtemperature in aqueous conditions with excellent regioselectivity(1,4 >1,5) by the addition of catalytic amounts of Cu(I) salts to thereaction mixture. See, e.g., Tornoe, et al., (2002) J. Org. Chem.67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem. Int. Ed.41:2596-2599. Another method that can be used is the ligand exchange ona bisarsenic compound with a tetracysteine motif, see, e.g., Griffin, etal., (1998) Science 281:269-272.

A molecule that can be added to a protein of the invention through a[3+2] cycloaddition includes virtually any molecule with an azide oralkynyl derivative. Molecules include, but are not limited to, dyes,fluorophores, crosslinking agents, saccharide derivatives, polymers(including but not limited to, derivatives of polyethylene glycol),photocrosslinkers, cytotoxic compounds, affinity labels, derivatives ofbiotin, resins, beads, a second protein or polypeptide (or more),polynucleotide(s) (including but not limited to, DNA, RNA, etc.), metalchelators, cofactors, fatty acids, carbohydrates, and the like. Thesemolecules can be added to an unnatural amino acid with an alkynyl group,including but not limited to, p-propargyloxyphenylalanine, or azidogroup, including but not limited to, p-azido-phenylalanine,respectively.

V. In Vivo Generation of FGF-21 Polypeptides ComprisingNon-Naturally-Encoded Amino Acids

The FGF-21 polypeptides of the invention can be generated in vivo usingmodified tRNA and tRNA synthetases to add to or substitute amino acidsthat are not encoded in naturally-occurring systems.

Methods for generating tRNAs and tRNA synthetases which use amino acidsthat are not encoded in naturally-occurring systems are described in,e.g., U.S. Pat. Nos. 7,045,337 and 7,083,970 which are incorporated byreference herein. These methods involve generating a translationalmachinery that functions independently of the synthetases and tRNAsendogenous to the translation system (and are therefore sometimesreferred to as “orthogonal”). Typically, the translation systemcomprises an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNAsynthetase (O—RS). Typically, the O—RS preferentially aminoacylates theO-tRNA with at least one non-naturally occurring amino acid in thetranslation system and the O-tRNA recognizes at least one selector codonthat is not recognized by other tRNAs in the system. The translationsystem thus inserts the non-naturally-encoded amino acid into a proteinproduced in the system, in response to an encoded selector codon,thereby “substituting” an amino acid into a position in the encodedpolypeptide.

A wide variety of orthogonal tRNAs and aminoacyl tRNA synthetases havebeen described in the art for inserting particular synthetic amino acidsinto polypeptides, and are generally suitable for use in the presentinvention. For example, keto-specific O-tRNA/aminoacyl-tRNA synthetasesare described in Wang, L., et al., Proc. Natl. Acad. Sci. USA 100:56-61(2003) and Zhang, Z. et al., Biochem. 42(22):6735-6746 (2003). ExemplaryO—RS, or portions thereof, are encoded by polynucleotide sequences andinclude amino acid sequences disclosed in U.S. Pat. Nos. 7,045,337 and7,083,970, each incorporated herein by reference. Corresponding O-tRNAmolecules for use with the O-RSs are also described in U.S. Pat. Nos.7,045,337 and 7,083,970 which are incorporated by reference herein.Additional examples of O-tRNA/aminoacyl-tRNA synthetase pairs aredescribed in WO 2005/007870, WO 2005/007624; and WO 2005/019415.

An example of an azide-specific O-tRNA/aminoacyl-tRNA synthetase systemis described in Chin, J. W., et al., J. Am. Chem. Soc. 124:9026-9027(2002). Exemplary O—RS sequences for p-azido-L-Phe include, but are notlimited to, nucleotide sequences SEQ ID NOs: 14-16 and 29-32 and aminoacid sequences SEQ ID NOs: 46-48 and 61-64 as disclosed in U.S. Pat. No.7,083,970 which is incorporated by reference herein. Exemplary O-tRNAsequences suitable for use in the present invention include, but are notlimited to, nucleotide sequences SEQ ID NOs: 1-3 as disclosed in U.S.Pat. No. 7,083,970) which is incorporated by reference herein. Otherexamples of O-tRNA/aminoacyl-tRNA synthetase pairs specific toparticular non-naturally encoded amino acids are described in U.S. Pat.No. 7,045,337 which is incorporated by reference herein. O—RS and O-tRNAthat incorporate both keto- and azide-containing amino acids in S.cerevisiae are described in Chin, J. W., et al., Science 301:964-967(2003).

Several other orthogonal pairs have been reported. Glutaminyl (see,e.g., Liu, D. R., and Schultz, P. G. (1999) Proc. Natl. Acad. Sci. U.S.A96:4780-4785), aspartyl (see, e.g., Pastrnak, M., et al., (2000) Helv.Chim. Acta 83:2277-2286), and tyrosyl (see, e.g., Ohno, S., et al.,(1998) J. Biochem. (Tokyo, Jpn.) 124:1065-1068; and, Kowal, A. K., etal., (2001) Proc. Natl. Acad. Sci. U.S.A 98:2268-2273) systems derivedfrom S. cerevisiae tRNA's and synthetases have been described for thepotential incorporation of unnatural amino acids in E. coli. Systemsderived from the E. coli glutaminyl (see, e.g., Kowal, A. K., et al.,(2001) Proc. Natl. Acad. Sci. U.S.A 98:2268-2273) and tyrosyl (see,e.g., Edwards, H., and Schimmel, P. (1990) Mol. Cell. Biol.10:1633-1641) synthetases have been described for use in S. cerevisiae.The E. coli tyrosyl system has been used for the incorporation of3-iodo-L-tyrosine in vivo, in mammalian cells. See, Sakamoto, K., etal., (2002) Nucleic Acids Res. 30:4692-4699.

Use of O-tRNA/aminoacyl-tRNA synthetases involves selection of aspecific codon which encodes the non-naturally encoded amino acid. Whileany codon can be used, it is generally desirable to select a codon thatis rarely or never used in the cell in which the O-tRNA/aminoacyl-tRNAsynthetase is expressed. For example, exemplary codons include nonsensecodon such as stop codons (amber, ochre, and opal), four or more basecodons and other natural three-base codons that are rarely or unused.

Specific selector codon(s) can be introduced into appropriate positionsin the FGF-21 polynucleotide coding sequence using mutagenesis methodsknown in the art (including but not limited to, site-specificmutagenesis, cassette mutagenesis, restriction selection mutagenesis,etc.).

Methods for generating components of the protein biosynthetic machinery,such as O-RSs, O-tRNAs, and orthogonal O-tRNA/O—RS pairs that can beused to incorporate a non-naturally encoded amino acid are described inWang, L., et al., Science 292: 498-500 (2001); Chin, J. W., et al., J.Am. Chem. Soc. 124:9026-9027 (2002); Zhang, Z. et al., Biochemistry 42:6735-6746 (2003). Methods and compositions for the in vivo incorporationof non-naturally encoded amino acids are described in U.S. Pat. No.7,045,337, which is incorporated by reference herein. Methods forselecting an orthogonal tRNA-tRNA synthetase pair for use in in vivotranslation system of an organism are also described in U.S. Pat. Nos.7,045,337 and 7,083,970 which are incorporated by reference herein. PCTPublication No. WO 04/035743 entitled “Site Specific Incorporation ofKeto Amino Acids into Proteins,” which is incorporated by referenceherein in its entirety, describes orthogonal RS and tRNA pairs for theincorporation of keto amino acids. PCT Publication No. WO 04/094593entitled “Expanding the Eukaryotic Genetic Code,” which is incorporatedby reference herein in its entirety, describes orthogonal RS and tRNApairs for the incorporation of non-naturally encoded amino acids ineukaryotic host cells.

Methods for producing at least one recombinant orthogonal aminoacyl-tRNAsynthetase (O—RS) comprise: (a) generating a library of (optionallymutant) RSs derived from at least one aminoacyl-tRNA synthetase (RS)from a first organism, including but not limited to, a prokaryoticorganism, such as Methanococcus jannaschii, Methanobacteriumthermoautotrophicum, Halobacterium, Escherichia coli, A. fulgidus, P.furiosus, P. horikoshii, A. pernix, T thermophilus, or the like, or aeukaryotic organism; (b) selecting (and/or screening) the library of RSs(optionally mutant RSs) for members that aminoacylate an orthogonal tRNA(O-tRNA) in the presence of a non-naturally encoded amino acid and anatural amino acid, thereby providing a pool of active (optionallymutant) RSs; and/or, (c) selecting (optionally through negativeselection) the pool for active RSs (including but not limited to, mutantRSs) that preferentially aminoacylate the O-tRNA in the absence of thenon-naturally encoded amino acid, thereby providing the at least onerecombinant 0-RS; wherein the at least one recombinant 0-RSpreferentially aminoacylates the O-tRNA with the non-naturally encodedamino acid.

In one embodiment, the RS is an inactive RS. The inactive RS can begenerated by mutating an active RS. For example, the inactive RS can begenerated by mutating at least about 1, at least about 2, at least about3, at least about 4, at least about 5, at least about 6, or at leastabout 10 or more amino acids to different amino acids, including but notlimited to, alanine.

Libraries of mutant RSs can be generated using various techniques knownin the art, including but not limited to rational design based onprotein three dimensional RS structure, or mutagenesis of RS nucleotidesin a random or rational design technique. For example, the mutant RSscan be generated by site-specific mutations, random mutations, diversitygenerating recombination mutations, chimeric constructs, rational designand by other methods described herein or known in the art.

In one embodiment, selecting (and/or screening) the library of RSs(optionally mutant RSs) for members that are active, including but notlimited to, that aminoacylate an orthogonal tRNA (O-tRNA) in thepresence of a non-naturally encoded amino acid and a natural amino acid,includes: introducing a positive selection or screening marker,including but not limited to, an antibiotic resistance gene, or thelike, and the library of (optionally mutant) RSs into a plurality ofcells, wherein the positive selection and/or screening marker comprisesat least one selector codon, including but not limited to, an amber,ochre, or opal codon; growing the plurality of cells in the presence ofa selection agent; identifying cells that survive (or show a specificresponse) in the presence of the selection and/or screening agent bysuppressing the at least one selector codon in the positive selection orscreening marker, thereby providing a subset of positively selectedcells that contains the pool of active (optionally mutant) RSs.Optionally, the selection and/or screening agent concentration can bevaried.

In one aspect, the positive selection marker is a chloramphenicolacetyltransferase (CAT) gene and the selector codon is an amber stopcodon in the CAT gene. Optionally, the positive selection marker is aβ-lactamase gene and the selector codon is an amber stop codon in theβ-lactamase gene. In another aspect the positive screening markercomprises a fluorescent or luminescent screening marker or an affinitybased screening marker (including but not limited to, a cell surfacemarker).

In one embodiment, negatively selecting or screening the pool for activeRSs (optionally mutants) that preferentially aminoacylate the O-tRNA inthe absence of the non-naturally encoded amino acid includes:introducing a negative selection or screening marker with the pool ofactive (optionally mutant) RSs from the positive selection or screeninginto a plurality of cells of a second organism, wherein the negativeselection or screening marker comprises at least one selector codon(including but not limited to, an antibiotic resistance gene, includingbut not limited to, a chloramphenicol acetyltransferase (CAT) gene);and, identifying cells that survive or show a specific screeningresponse in a first medium supplemented with the non-naturally encodedamino acid and a screening or selection agent, but fail to survive or toshow the specific response in a second medium not supplemented with thenon-naturally encoded amino acid and the selection or screening agent,thereby providing surviving cells or screened cells with the at leastone recombinant O—RS. For example, a CAT identification protocoloptionally acts as a positive selection and/or a negative screening indetermination of appropriate O—RS recombinants. For instance, a pool ofclones is optionally replicated on growth plates containing CAT (whichcomprises at least one selector codon) either with or without one ormore non-naturally encoded amino acid. Colonies growing exclusively onthe plates containing non-naturally encoded amino acids are thusregarded as containing recombinant O—RS. In one aspect, theconcentration of the selection (and/or screening) agent is varied. Insome aspects the first and second organisms are different. Thus, thefirst and/or second organism optionally comprises: a prokaryote, aeukaryote, a mammal, an Escherichia coli, a fungi, a yeast, anarchaebacterium, a eubacterium, a plant, an insect, a protist, etc. Inother embodiments, the screening marker comprises a fluorescent orluminescent screening marker or an affinity based screening marker.

In another embodiment, screening or selecting (including but not limitedto, negatively selecting) the pool for active (optionally mutant) RSsincludes: isolating the pool of active mutant RSs from the positiveselection step (b); introducing a negative selection or screeningmarker, wherein the negative selection or screening marker comprises atleast one selector codon (including but not limited to, a toxic markergene, including but not limited to, a ribonuclease barnase gene,comprising at least one selector codon), and the pool of active(optionally mutant) RSs into a plurality of cells of a second organism;and identifying cells that survive or show a specific screening responsein a first medium not supplemented with the non-naturally encoded aminoacid, but fail to survive or show a specific screening response in asecond medium supplemented with the non-naturally encoded amino acid,thereby providing surviving or screened cells with the at least onerecombinant O—RS, wherein the at least one recombinant O—RS is specificfor the non-naturally encoded amino acid. In one aspect, the at leastone selector codon comprises about two or more selector codons. Suchembodiments optionally can include wherein the at least one selectorcodon comprises two or more selector codons, and wherein the first andsecond organism are different (including but not limited to, eachorganism is optionally, including but not limited to, a prokaryote, aeukaryote, a mammal, an Escherichia coli, a fungi, a yeast, anarchaebacteria, a eubacteria, a plant, an insect, a protist, etc.).Also, some aspects include wherein the negative selection markercomprises a ribonuclease barnase gene (which comprises at least oneselector codon). Other aspects include wherein the screening markeroptionally comprises a fluorescent or luminescent screening marker or anaffinity based screening marker. In the embodiments herein, thescreenings and/or selections optionally include variation of thescreening and/or selection stringency.

In one embodiment, the methods for producing at least one recombinantorthogonal aminoacyl-tRNA synthetase (O—RS) can further comprise: (d)isolating the at least one recombinant O—RS; (e) generating a second setof O—RS (optionally mutated) derived from the at least one recombinantO—RS; and, (f) repeating steps (b) and (c) until a mutated O—RS isobtained that comprises an ability to preferentially aminoacylate theO-tRNA. Optionally, steps (d)-(f) are repeated, including but notlimited to, at least about two times. In one aspect, the second set ofmutated O—RS derived from at least one recombinant O—RS can be generatedby mutagenesis, including but not limited to, random mutagenesis,site-specific mutagenesis, recombination or a combination thereof.

The stringency of the selection/screening steps, including but notlimited to, the positive selection/screening step (b), the negativeselection/screening step (c) or both the positive and negativeselection/screening steps (b) and (c), in the above-described methods,optionally includes varying the selection/screening stringency. Inanother embodiment, the positive selection/screening step (b), thenegative selection/screening step (c) or both the positive and negativeselection/screening steps (b) and (c) comprise using a reporter, whereinthe reporter is detected by fluorescence-activated cell sorting (FACS)or wherein the reporter is detected by luminescence. Optionally, thereporter is displayed on a cell surface, on a phage display or the likeand selected based upon affinity or catalytic activity involving thenon-naturally encoded amino acid or an analogue. In one embodiment, themutated synthetase is displayed on a cell surface, on a phage display orthe like.

Methods for producing a recombinant orthogonal tRNA (O-tRNA) include:(a) generating a library of mutant tRNAs derived from at least one tRNA,including but not limited to, a suppressor tRNA, from a first organism;(b) selecting (including but not limited to, negatively selecting) orscreening the library for (optionally mutant) tRNAs that areaminoacylated by an aminoacyl-tRNA synthetase (RS) from a secondorganism in the absence of a RS from the first organism, therebyproviding a pool of tRNAs (optionally mutant); and, (c) selecting orscreening the pool of tRNAs (optionally mutant) for members that areaminoacylated by an introduced orthogonal RS (O—RS), thereby providingat least one recombinant O-tRNA; wherein the at least one recombinantO-tRNA recognizes a selector codon and is not efficiency recognized bythe RS from the second organism and is preferentially aminoacylated bythe O—RS. In some embodiments the at least one tRNA is a suppressor tRNAand/or comprises a unique three base codon of natural and/or unnaturalbases, or is a nonsense codon, a rare codon, an unnatural codon, a codoncomprising at least 4 bases, an amber codon, an ochre codon, or an opalstop codon. In one embodiment, the recombinant O-tRNA possesses animprovement of orthogonality. It will be appreciated that in someembodiments, O-tRNA is optionally imported into a first organism from asecond organism without the need for modification. In variousembodiments, the first and second organisms are either the same ordifferent and are optionally chosen from, including but not limited to,prokaryotes (including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Escherichia coli, Halobacterium,etc.), eukaryotes, mammals, fungi, yeasts, archaebacteria, eubacteria,plants, insects, protists, etc. Additionally, the recombinant tRNA isoptionally aminoacylated by a non-naturally encoded amino acid, whereinthe non-naturally encoded amino acid is biosynthesized in vivo eithernaturally or through genetic manipulation. The non-naturally encodedamino acid is optionally added to a growth medium for at least the firstor second organism.

In one aspect, selecting (including but not limited to, negativelyselecting) or screening the library for (optionally mutant) tRNAs thatare aminoacylated by an aminoacyl-tRNA synthetase (step (b)) includes:introducing a toxic marker gene, wherein the toxic marker gene comprisesat least one of the selector codons (or a gene that leads to theproduction of a toxic or static agent or a gene essential to theorganism wherein such marker gene comprises at least one selector codon)and the library of (optionally mutant) tRNAs into a plurality of cellsfrom the second organism; and, selecting surviving cells, wherein thesurviving cells contain the pool of (optionally mutant) tRNAs comprisingat least one orthogonal tRNA or nonfunctional tRNA. For example,surviving cells can be selected by using a comparison ratio cell densityassay.

In another aspect, the toxic marker gene can include two or moreselector codons. In another embodiment of the methods, the toxic markergene is a ribonuclease barnase gene, where the ribonuclease barnase genecomprises at least one amber codon. Optionally, the ribonuclease barnasegene can include two or more amber codons.

In one embodiment, selecting or screening the pool of (optionallymutant) tRNAs for members that are aminoacylated by an introducedorthogonal RS (O—RS) can include: introducing a positive selection orscreening marker gene, wherein the positive marker gene comprises a drugresistance gene (including but not limited to, β-lactamase gene,comprising at least one of the selector codons, such as at least oneamber stop codon) or a gene essential to the organism, or a gene thatleads to detoxification of a toxic agent, along with the O—RS, and thepool of (optionally mutant) tRNAs into a plurality of cells from thesecond organism; and, identifying surviving or screened cells grown inthe presence of a selection or screening agent, including but notlimited to, an antibiotic, thereby providing a pool of cells possessingthe at least one recombinant tRNA, where the at least one recombinanttRNA is aminoacylated by the O—RS and inserts an amino acid into atranslation product encoded by the positive marker gene, in response tothe at least one selector codons. In another embodiment, theconcentration of the selection and/or screening agent is varied.

Methods for generating specific O-tRNA/O—RS pairs are provided. Methodsinclude: (a) generating a library of mutant tRNAs derived from at leastone tRNA from a first organism; (b) negatively selecting or screeningthe library for (optionally mutant) tRNAs that are aminoacylated by anaminoacyl-tRNA synthetase (RS) from a second organism in the absence ofa RS from the first organism, thereby providing a pool of (optionallymutant) tRNAs; (c) selecting or screening the pool of (optionallymutant) tRNAs for members that are aminoacylated by an introducedorthogonal RS (O—RS), thereby providing at least one recombinant O-tRNA.The at least one recombinant 0-tRNA recognizes a selector codon and isnot efficiency recognized by the RS from the second organism and ispreferentially aminoacylated by the 0-RS. The method also includes (d)generating a library of (optionally mutant) RSs derived from at leastone aminoacyl-tRNA synthetase (RS) from a third organism; (e) selectingor screening the library of mutant RSs for members that preferentiallyaminoacylate the at least one recombinant O-tRNA in the presence of anon-naturally encoded amino acid and a natural amino acid, therebyproviding a pool of active (optionally mutant) RSs; and, (f) negativelyselecting or screening the pool for active (optionally mutant) RSs thatpreferentially aminoacylate the at least one recombinant O-tRNA in theabsence of the non-naturally encoded amino acid, thereby providing theat least one specific O-tRNA/O—RS pair, wherein the at least onespecific O-tRNA/O—RS pair comprises at least one recombinant 0-RS thatis specific for the non-naturally encoded amino acid and the at leastone recombinant O-tRNA. Specific O-tRNA/O—RS pairs produced by themethods are included. For example, the specific 0-tRNA/O—RS pair caninclude, including but not limited to, a mutRNATyr-mutTyrRS pair, suchas a mutRNATyr-SS12TyrRS pair, a mutRNALeu-mutLeuRS pair, amutRNAThr-mutThrRS pair, a mutRNAGlu-mutGluRS pair, or the like.Additionally, such methods include wherein the first and third organismare the same (including but not limited to, Methanococcus jannaschii).

Methods for selecting an orthogonal tRNA-tRNA synthetase pair for use inan in vivo translation system of a second organism are also included inthe present invention. The methods include: introducing a marker gene, atRNA and an aminoacyl-tRNA synthetase (RS) isolated or derived from afirst organism into a first set of cells from the second organism;introducing the marker gene and the tRNA into a duplicate cell set froma second organism; and, selecting for surviving cells in the first setthat fail to survive in the duplicate cell set or screening for cellsshowing a specific screening response that fail to give such response inthe duplicate cell set, wherein the first set and the duplicate cell setare grown in the presence of a selection or screening agent, wherein thesurviving or screened cells comprise the orthogonal tRNA-tRNA synthetasepair for use in the in the in vivo translation system of the secondorganism. In one embodiment, comparing and selecting or screeningincludes an in vivo complementation assay. The concentration of theselection or screening agent can be varied.

The organisms of the present invention comprise a variety of organismand a variety of combinations. For example, the first and the secondorganisms of the methods of the present invention can be the same ordifferent. In one embodiment, the organisms are optionally a prokaryoticorganism, including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli,A. fulgidus, P. furiosus, P. horikoshii, A. pernix, T thermophilus, orthe like. Alternatively, the organisms optionally comprise a eukaryoticorganism, including but not limited to, plants (including but notlimited to, complex plants such as monocots, or dicots), algae,protists, fungi (including but not limited to, yeast, etc), animals(including but not limited to, mammals, insects, arthropods, etc.), orthe like. In another embodiment, the second organism is a prokaryoticorganism, including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli,A. fulgidus, Halobacterium, P. furiosus, P. horikoshii, A. pernix, Tthermophilus, or the like. Alternatively, the second organism can be aeukaryotic organism, including but not limited to, a yeast, a animalcell, a plant cell, a fungus, a mammalian cell, or the like. In variousembodiments the first and second organisms are different.

VI. Location of Non-Naturally-Occurring Amino Acids in FGF-21Polypeptides

The present invention contemplates incorporation of one or morenon-naturally-occurring amino acids into FGF-21 polypeptides. One ormore non-naturally-occurring amino acids may be incorporated at aparticular position which does not disrupt activity of the polypeptide.This can be achieved by making “conservative” substitutions, includingbut not limited to, substituting hydrophobic amino acids withhydrophobic amino acids, bulky amino acids for bulky amino acids,hydrophilic amino acids for hydrophilic amino acids and/or inserting thenon-naturally-occurring amino acid in a location that is not requiredfor activity.

A variety of biochemical and structural approaches can be employed toselect the desired sites for substitution with a non-naturally encodedamino acid within the FGF-21 polypeptide. It is readily apparent tothose of ordinary skill in the art that any position of the polypeptidechain is suitable for selection to incorporate a non-naturally encodedamino acid, and selection may be based on rational design or by randomselection for any or no particular desired purpose. Selection of desiredsites may be for producing an FGF-21 molecule having any desiredproperty or activity, including but not limited to, agonists,super-agonists, inverse agonists, antagonists, receptor bindingmodulators, receptor activity modulators, dimer or multimer formation,no change to activity or property compared to the native molecule, ormanipulating any physical or chemical property of the polypeptide suchas solubility, aggregation, or stability. For example, locations in thepolypeptide required for biological activity of FGF-21 polypeptides canbe identified using point mutation analysis, alanine scanning,saturation mutagenesis and screening for biological activity, or homologscanning methods known in the art. Residues that are critical for FGF-21bioactivity, residues that are involved with pharmaceutical stability,antibody epitopes, or receptor or heparin binding residues may bemutated. U.S. Pat. Nos. 5,580,723; 5,834,250; 6,013,478; 6,428,954; and6,451,561, which are incorporated by reference herein, describe methodsfor the systematic analysis of the structure and function ofpolypeptides such as FGF-21 by identifying active domains whichinfluence the activity of the polypeptide with a target substance.Residues other than those identified as critical to biological activityby alanine or homolog scanning mutagenesis may be good candidates forsubstitution with a non-naturally encoded amino acid depending on thedesired activity sought for the polypeptide. Alternatively, the sitesidentified as critical to biological activity may also be goodcandidates for substitution with a non-naturally encoded amino acid,again depending on the desired activity sought for the polypeptide.Another alternative would be to simply make serial substitutions in eachposition on the polypeptide chain with a non-naturally encoded aminoacid and observe the effect on the activities of the polypeptide. It isreadily apparent to those of ordinary skill in the art that any means,technique, or method for selecting a position for substitution with anon-natural amino acid into any polypeptide is suitable for use in thepresent invention.

A lot of data has already been collected using the methods presented inthis application and potential and beneficial sites of mutation havebeen found and successfully tested, as described in the examplesprovided later in this specification. Finding additional informationregarding structure and activity of mutants, even those which includesome details regarding formulation and/or testing that has not beenspecifically described in the examples, of FGF-21 polypeptides thatcontain deletions can also be examined to determine regions of theprotein that are likely to be tolerant of substitution with anon-naturally encoded amino acid. In a similar manner, proteasedigestion and monoclonal antibodies can be used to identify regions ofFGF-21 that are responsible for binding the FGF-21 receptor. Onceresidues that are likely to be intolerant to substitution withnon-naturally encoded amino acids have been eliminated, the impact ofproposed substitutions at each of the remaining positions can beexamined. Models may be generated from the three-dimensional crystalstructures of other FGF family members and FGF receptors. Protein DataBank (PDB, available on the World Wide Web at rcsb.org) is a centralizeddatabase containing three-dimensional structural data of large moleculesof proteins and nucleic acids. Models may be made investigating thesecondary and tertiary structure of polypeptides, if three-dimensionalstructural data is not available. Thus, those of ordinary skill in theart can readily identify amino acid positions that can be substitutedwith non-naturally encoded amino acids.

In some embodiments, the FGF-21 polypeptides of the invention compriseone or more non-naturally occurring amino acids positioned in a regionof the protein that does not disrupt the structure of the polypeptide.

Exemplary residues of incorporation of a non-naturally encoded aminoacid may be those that are excluded from potential receptor bindingregions, may be fully or partially solvent exposed, have minimal or nohydrogen-bonding interactions with nearby residues, may be minimallyexposed to nearby reactive residues, may be on one or more of theexposed faces, may be a site or sites that are juxtaposed to a secondFGF-21, or other molecule or fragment thereof, may be in regions thatare highly flexible, or structurally rigid, as predicted by thethree-dimensional, secondary, tertiary, or quaternary structure ofFGF-21, bound or unbound to its receptor, or coupled or not coupled toanother biologically active molecule, or may modulate the conformationof the FGF-21 itself or a dimer or multimer comprising one or moreFGF-21, by altering the flexibility or rigidity of the completestructure as desired.

The family of FGF proteins have a common β-trefoil or β-sheet structureas identified by crystallography (Harmer et al., Biochemistry 43:629-640(2004)). One of ordinary skill in the art recognizes that such analysisof FGF-21 enables the determination of which amino acid residues aresurface exposed compared to amino acid residues that are buried withinthe tertiary structure of the protein. Therefore, it is an embodiment ofthe present invention to substitute a non-naturally encoded amino acidfor an amino acid that is a surface exposed residue.

In some embodiments, one or more non-naturally encoded amino acids areincorporated at any position in FGF-21: 1-181 from SEQ ID NO: 1 or thecorresponding amino acids in SEQ ID NOs: 2-7. In some embodiments, oneor more non-naturally encoded amino acids are incorporated in one ormore of the following positions in FGF-21: before position 1 (i.e. atthe N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182 (i.e., at the carboxyl terminusof the protein) (SEQ ID NO: 1 or the corresponding amino acids in SEQ IDNOs: 2-7). In some embodiments, one or more non-naturally encoded aminoacids are incorporated in one or more of the following positions inFGF-21: 10, 52, 117, 126, 131, 162, 87, 77, 83, 72, 69, 79, 91, 96, 108,and 110 (SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs:2-7). In some embodiments, one or more non-naturally encoded amino acidsare incorporated in one or more of the following positions in FGF-21:10, 52, 77, 117, 126, 131, and 162 (SEQ ID NO: 1 or the correspondingamino acids of SEQ ID NOs: 2-7). In some embodiments, one or morenon-naturally encoded amino acids are incorporated in one or more of thefollowing positions in FGF-21: 87, 77, 83, 72 (SEQ ID NO: 1 or thecorresponding amino acids of SEQ ID NOs: 2-7). In some embodiments, oneor more non-naturally encoded amino acids are incorporated in one ormore of the following positions in FGF-21: 69, 79, 91, 96, 108, and 110(SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs: 2-7).

In one embodiment, the non-naturally occurring amino acid is at the 91position in FGF-21 (SEQ ID NO: 1 or the corresponding amino acids of SEQID NOs: 2-7). In one embodiment, the non-naturally occurring amino acidis at the 131 position in FGF-21 (SEQ ID NO: 1 or the correspondingamino acids of SEQ ID NOs: 2-7). In one embodiment, the non-naturallyoccurring amino acid is at the 108 position in FGF-21 (SEQ ID NO: 1 orthe corresponding amino acids of SEQ ID NOs: 2-7). In one embodiment,the non-naturally occurring amino acid is at the 77 position in FGF-21(SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs: 2-7). Inone embodiment, the non-naturally occurring amino acid is at the 72position in FGF-21 (SEQ ID NO: 1 or the corresponding amino acids of SEQID NOs: 2-7). In one embodiment, the non-naturally occurring amino acidis at the 87 position in FGF-21 (SEQ ID NO: 1 or the corresponding aminoacids of SEQ ID NOs: 2-7). In one embodiment, the non-naturallyoccurring amino acid is at the 86 position in FGF-21 (SEQ ID NO: 1 orthe corresponding amino acids of SEQ ID NOs: 2-7). In one embodiment,the non-naturally occurring amino acid is at the 126 position in FGF-21(SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs: 2-7). Inone embodiment, the non-naturally occurring amino acid is at the 110position in FGF-21 (SEQ ID NO: 1 or the corresponding amino acids of SEQID NOs: 2-7). In one embodiment, the non-naturally occurring amino acidis at the 83 position in FGF-21 (SEQ ID NO: 1 or the corresponding aminoacids of SEQ ID NOs: 2-7). In one embodiment, the non-naturallyoccurring amino acid is at the 146 position in FGF-21 (SEQ ID NO: 1 orthe corresponding amino acids of SEQ ID NOs: 2-7). In one embodiment,the non-naturally occurring amino acid is at the 135 position in FGF-21(SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs: 2-7). Inone embodiment, the non-naturally occurring amino acid is at the 96position in FGF-21 (SEQ ID NO: 1 or the corresponding amino acids of SEQID NOs: 2-7). In one embodiment, the non-naturally occurring amino acidis at the 36 position in FGF-21 (SEQ ID NO: 1 or the corresponding aminoacids of SEQ ID NOs: 2-7).

In another embodiment, there is a non-naturally occurring amino acid at91 and one other non-naturally occurring amino acid at one of thefollowing positions: before position 1 (i.e. at the N-terminus), 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182 (i.e., at the carboxyl terminus of the protein) (SEQ IDNO: 1 or the corresponding amino acids in SEQ ID NOs: 2-7). In anotherembodiment, there is a non-naturally occurring amino acid at 91 and oneother non-naturally occurring amino acid at one of the followingpositions: 131, 108, 77, 72, 87, 86, 126, 110, 83, 146, 135, 96, and 36(SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs: 2-7). Inanother embodiment, there is a non-naturally occurring amino acid atposition 91 and position 131 (SEQ ID NO: 1 or the corresponding aminoacids in SEQ ID NOs: 2-7). In another embodiment, there is anon-naturally occurring amino acid at position 91 and position 77 (SEQID NO: 1 or the corresponding amino acids in SEQ ID NOs: 2-7). Inanother embodiment, there is a non-naturally occurring amino acid atposition 91 and position 108 (SEQ ID NO: 1 or the corresponding aminoacids in SEQ ID NOs: 2-7). In another embodiment, there is anon-naturally occurring amino acid at position 131 and position 108 (SEQID NO: 1 or the corresponding amino acids in SEQ ID NOs: 2-7). Inanother embodiment, there is a non-naturally occurring amino acid atposition 131 and position 77 (SEQ ID NO: 1 or the corresponding aminoacids in SEQ ID NOs: 2-7). In another embodiment, there is anon-naturally occurring amino acid at position 131 (SEQ ID NO: 1 or thecorresponding amino acids in SEQ ID NOs: 2-7). In another embodiment,there is a non-naturally occurring amino acid at position 108 (SEQ IDNO: 1 or the corresponding amino acids in SEQ ID NOs: 2-7). In anotherembodiment, there is a non-naturally occurring amino acid at position 77(SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs: 2-7). Inanother embodiment, there is a non-naturally occurring amino acid atposition 72 (SEQ ID NO: 1 or the corresponding amino acids in SEQ IDNOs: 2-7). In another embodiment, there is a non-naturally occurringamino acid at position 87 (SEQ ID NO: 1 or the corresponding amino acidsin SEQ ID NOs: 2-7). In another embodiment, there is a non-naturallyoccurring amino acid at position 86 (SEQ ID NO: 1 or the correspondingamino acids in SEQ ID NOs: 2-7) linked. In another embodiment, there isa non-naturally occurring amino acid at position 126 (SEQ ID NO: 1 orthe corresponding amino acids in SEQ ID NOs: 2-7). In anotherembodiment, there is a non-naturally occurring amino acid at position110 (SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs: 2-7).In another embodiment, there is a non-naturally occurring amino acid atposition 83 (SEQ ID NO: 1 or the corresponding amino acids in SEQ IDNOs: 2-7).

In another embodiment, there is a non-naturally occurring amino acid at91 and one or more other non-naturally occurring amino acids at one ormore of the following positions: before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182 (i.e., at the carboxyl terminus of theprotein) (SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs:2-7). In another embodiment, there is a non-naturally occurring aminoacid at 91 and one or more other non-naturally occurring amino acid atone or more of the following positions: 131, 108, 77, 72, 87, 86, 126,110, 83, 146, 135, 96, and 36 (SEQ ID NO: 1 or the corresponding aminoacids of SEQ ID NOs: 2-7).

In another embodiment, there is a non-naturally occurring amino acid at131 and one other non-naturally occurring amino acid at one or more ofthe following positions: before position 1 (i.e. at the N-terminus), 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182 (i.e., at the carboxyl terminus of the protein) (SEQID NO: 1 or the corresponding amino acids in SEQ ID NOs: 2-7). Inanother embodiment, there is a non-naturally occurring amino acid at 131and one other non-naturally occurring amino acid at one or more of thefollowing positions: 131, 108, 77, 72, 87, 86, 126, 110, 83, 146, 135,96, and 36 (SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs:2-7).

In another embodiment, there is a non-naturally occurring amino acid atposition 108 and two or more other non-naturally occurring amino acidsat two or more of the following positions: before position 1 (i.e. atthe N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182 (i.e., at the carboxyl terminusof the protein) (SEQ ID NO: 1 or the corresponding amino acids in SEQ IDNOs: 2-7). In another embodiment, there is a non-naturally occurringamino acid at position 108 and two or more other non-naturally occurringamino acids at two or more of the following positions: 131, 108, 77, 72,87, 86, 126, 110, 83, 146, 135, 96, and 36 (SEQ ID NO: 1 or thecorresponding amino acids of SEQ ID NOs: 2-7).

In another embodiment, there is a non-naturally occurring amino acid at77 and one other non-naturally occurring amino acid at one or more ofthe following positions: before position 1 (i.e. at the N-terminus), 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182 (i.e., at the carboxyl terminus of the protein) (SEQID NO: 1 or the corresponding amino acids in SEQ ID NOs: 2-7). Inanother embodiment, there is a non-naturally occurring amino acid at 77and one other non-naturally occurring amino acid at one or more of thefollowing positions: 131, 108, 77, 72, 87, 86, 126, 110, 83, 146, 135,96, and 36 (SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs:2-7).

An examination of the crystal structure of FGF-21 or FGF familymember(s) and its interaction with the FGF receptor can indicate whichcertain amino acid residues have side chains that are fully or partiallyaccessible to solvent. The side chain of a non-naturally encoded aminoacid at these positions may point away from the protein surface and outinto the solvent. In some embodiments, the non-naturally occurring aminoacid at one or more of these positions is linked to a water solublepolymer, including but not limited to, positions: before position 1(i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,174, 175, 176, 177, 178, 179, 180, 181, 182 (i.e., at the carboxylterminus of the protein) (SEQ ID NO: 1 or the corresponding amino acidsin SEQ ID NOs: 2-7). In some embodiments, the non-naturally occurringamino acid at one of these positions is linked to a water solublepolymer, including positions: before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182 (i.e., at the carboxyl terminus of theprotein) (SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs:2-7). In some embodiments, the non-naturally occurring amino acid at twoor more of these positions is linked to a water soluble polymer,including but not limited to, positions: before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182 (i.e., at the carboxyl terminus of theprotein) (SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs:2-7). In some embodiments, the non-naturally occurring amino acid at oneor more of these positions is linked to a water soluble polymer,including but not limited to, positions: 10, 52, 117, 126, 131, 162, 87,77, 83, 72, 69, 79, 91, 96, 108, and 110 (SEQ ID NO: 1 or thecorresponding amino acids of SEQ ID NOs: 2-7). In some embodiments, thenon-naturally occurring amino acid at one or more of these positions islinked to a water soluble polymer: 10, 52, 77, 117, 126, 131, 162, (SEQID NO: 1 or the corresponding amino acids of SEQ ID NOs: 2-7). In someembodiments, the non-naturally occurring amino acid at one or more ofthese positions is linked to a water soluble polymer: 87, 77, 83, 72(SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs: 2-7). Insome embodiments, the non-naturally occurring amino acid at one or moreof these positions is linked to a water soluble polymer: 69, 79, 91, 96,108, and 110 (SEQ ID NO: 1 or the corresponding amino acids of SEQ IDNOs: 2-7). In some embodiments, the non-naturally occurring amino acidat one or more of these positions is linked to a water soluble polymer:91, 131, 108, 77, 72, 87, 86, 126, 110, 83, 146, 135, 96, and 36 (SEQ IDNO: 1 or the corresponding amino acids of SEQ ID NOs: 2-7). In anotherembodiment, where a non-naturally occurring amino acid occurs at aminoacid 91 (SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs:2-7) the non-naturally occurring amino acid is linked to a water solublepolymer.

In another embodiment, there is a non-naturally occurring amino acid at91 linked to a water soluble polymer and one other non-naturallyoccurring amino acid at one of the following positions and thesenon-naturally occurring amino acids are linked to a water solublepolymer: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182(i.e., at the carboxyl terminus of the protein) (SEQ ID NO: 1 or thecorresponding amino acids in SEQ ID NOs: 2-7). In another embodiment,there is a non-naturally occurring amino acid at 91 and one or moreother non-naturally occurring amino acid at one of the followingpositions and these non-naturally occurring amino acids are linked to awater soluble polymer: 131, 108, 77, 72, 87, 86, 126, 110, 83, 146, 135,96, and 36 (SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs:2-7). In another embodiment, there is a non-naturally occurring aminoacid at 91 linked to a water soluble polymer and one or more othernon-naturally occurring amino acid at one of the following positions andthese non-naturally occurring amino acids are linked to a water solublepolymer: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182(i.e., at the carboxyl terminus of the protein) (SEQ ID NO: 1 or thecorresponding amino acids in SEQ ID NOs: 2-7). In another embodiment,there is a non-naturally occurring amino acid at 91 linked to a watersoluble polymer and one or more other non-naturally occurring aminoacids which are linked to a water soluble polymer: 131, 108, 77, 72, 87,86, 126, 110, 83, 146, 135, 96, and 36 (SEQ ID NO: 1 or thecorresponding amino acids of SEQ ID NOs: 2-7). In another embodiment,there is a non-naturally occurring amino acid at 91 linked to a watersoluble polymer and two or more other non-naturally occurring amino acidat two or more of the following positions and these non-naturallyoccurring amino acids are linked to a water soluble polymer: beforeposition 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 (i.e., at thecarboxyl terminus of the protein) (SEQ ID NO: 1 or the correspondingamino acids in SEQ ID NOs: 2-7). In another embodiment, there is anon-naturally occurring amino acid at 91 linked to a water solublepolymer and two or more other non-naturally occurring amino acid at twoor more of the following positions and these non-naturally occurringamino acids are linked to a water soluble polymer: 131, 108, 77, 72, 87,86, 126, 110, 83, 146, 135, 96, and 36 (SEQ ID NO: 1 or thecorresponding amino acids of SEQ ID NOs: 2-7).

In another embodiment, there is a non-naturally occurring amino acid at131 linked to a water soluble polymer and one other non-naturallyoccurring amino acid at one of the following positions and thesenon-naturally occurring amino acids are linked to a water solublepolymer: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 (i.e.,at the carboxyl terminus of the protein) (SEQ ID NO: 1 or thecorresponding amino acids in SEQ ID NOs: 2-7). In another embodiment,there is a non-naturally occurring amino acid at 131 and one or moreother non-naturally occurring amino acid at one of the followingpositions and these non-naturally occurring amino acids are linked to awater soluble polymer: 91, 108, 77, 72, 87, 86, 126, 110, 83, 146, 135,96, and 36 (SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs:2-7). In another embodiment, there is a non-naturally occurring aminoacid at 131 linked to a water soluble polymer and one or more othernon-naturally occurring amino acid at one of the following positions andthese non-naturally occurring amino acids are linked to a water solublepolymer: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 (i.e.,at the carboxyl terminus of the protein) (SEQ ID NO: 1 or thecorresponding amino acids in SEQ ID NOs: 2-7). In another embodiment,there is a non-naturally occurring amino acid at 131 linked to a watersoluble polymer and one or more other non-naturally occurring aminoacids which are linked to a water soluble polymer: 91, 108, 77, 72, 87,86, 126, 110, 83, 146, 135, 96, and 36 (SEQ ID NO: 1 or thecorresponding amino acids of SEQ ID NOs: 2-7). In another embodiment,there is a non-naturally occurring amino acid at 131 linked to a watersoluble polymer and two or more other non-naturally occurring amino acidat two or more of the following positions and these non-naturallyoccurring amino acids are linked to a water soluble polymer: beforeposition 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 (i.e., at thecarboxyl terminus of the protein) (SEQ ID NO: 1 or the correspondingamino acids in SEQ ID NOs: 2-7). In another embodiment, there is anon-naturally occurring amino acid at 131 linked to a water solublepolymer and two or more other non-naturally occurring amino acid at twoor more of the following positions and these non-naturally occurringamino acids are linked to a water soluble polymer: 91, 108, 77, 72, 87,86, 126, 110, 83, 146, 135, 96, and 36 (SEQ ID NO: 1 or thecorresponding amino acids of SEQ ID NOs: 2-7).

In another embodiment, there is a non-naturally occurring amino acid at108 linked to a water soluble polymer and one other non-naturallyoccurring amino acid at one of the following positions and thesenon-naturally occurring amino acids are linked to a water solublepolymer: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 (i.e.,at the carboxyl terminus of the protein) (SEQ ID NO: 1 or thecorresponding amino acids in SEQ ID NOs: 2-7). In another embodiment,there is a non-naturally occurring amino acid at 108 and one or moreother non-naturally occurring amino acid at one of the followingpositions and these non-naturally occurring amino acids are linked to awater soluble polymer: 91, 131, 77, 72, 87, 86, 126, 110, 83, 146, 135,96, and 36 (SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs:2-7). In another embodiment, there is a non-naturally occurring aminoacid at 108 linked to a water soluble polymer and one or more othernon-naturally occurring amino acid at one of the following positions andthese non-naturally occurring amino acids are linked to a water solublepolymer: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 (i.e.,at the carboxyl terminus of the protein) (SEQ ID NO: 1 or thecorresponding amino acids in SEQ ID NOs: 2-7). In another embodiment,there is a non-naturally occurring amino acid at 108 linked to a watersoluble polymer and one or more other non-naturally occurring aminoacids which are linked to a water soluble polymer: 91, 131, 77, 72, 87,86, 126, 110, 83, 146, 135, 96, and 36 (SEQ ID NO: 1 or thecorresponding amino acids of SEQ ID NOs: 2-7). In another embodiment,there is a non-naturally occurring amino acid at 108 linked to a watersoluble polymer and two or more other non-naturally occurring amino acidat two or more of the following positions and these non-naturallyoccurring amino acids are linked to a water soluble polymer: beforeposition 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 (i.e., at thecarboxyl terminus of the protein) (SEQ ID NO: 1 or the correspondingamino acids in SEQ ID NOs: 2-7). In another embodiment, there is anon-naturally occurring amino acid at 108 linked to a water solublepolymer and two or more other non-naturally occurring amino acid at twoor more of the following positions and these non-naturally occurringamino acids are linked to a water soluble polymer: 91, 131, 77, 72, 87,86, 126, 110, 83, 146, 135, 96, and 36 (SEQ ID NO: 1 or thecorresponding amino acids of SEQ ID NOs: 2-7).

In another embodiment, there is a non-naturally occurring amino acid at77 linked to a water soluble polymer and one other non-naturallyoccurring amino acid at one of the following positions and thesenon-naturally occurring amino acids are linked to a water solublepolymer: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182(i.e., at the carboxyl terminus of the protein) (SEQ ID NO: 1 or thecorresponding amino acids in SEQ ID NOs: 2-7). In another embodiment,there is a non-naturally occurring amino acid at 77 and one or moreother non-naturally occurring amino acid at one of the followingpositions and these non-naturally occurring amino acids are linked to awater soluble polymer: 91, 131, 108, 72, 87, 86, 126, 110, 83, 146, 135,96, and 36 (SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs:2-7). In another embodiment, there is a non-naturally occurring aminoacid at 77 linked to a water soluble polymer and one or more othernon-naturally occurring amino acid at one of the following positions andthese non-naturally occurring amino acids are linked to a water solublepolymer: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182(i.e., at the carboxyl terminus of the protein) (SEQ ID NO: 1 or thecorresponding amino acids in SEQ ID NOs: 2-7). In another embodiment,there is a non-naturally occurring amino acid at 77 linked to a watersoluble polymer and one or more other non-naturally occurring aminoacids which are linked to a water soluble polymer: 91, 131, 108, 72, 87,86, 126, 110, 83, 146, 135, 96, and 36 (SEQ ID NO: 1 or thecorresponding amino acids of SEQ ID NOs: 2-7). In another embodiment,there is a non-naturally occurring amino acid at 77 linked to a watersoluble polymer and two or more other non-naturally occurring amino acidat two or more of the following positions and these non-naturallyoccurring amino acids are linked to a water soluble polymer: beforeposition 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 (i.e., at thecarboxyl terminus of the protein) (SEQ ID NO: 1 or the correspondingamino acids in SEQ ID NOs: 2-7). In another embodiment, there is anon-naturally occurring amino acid at 77 linked to a water solublepolymer and two or more other non-naturally occurring amino acid at twoor more of the following positions and these non-naturally occurringamino acids are linked to a water soluble polymer: 91, 131, 108, 72, 87,86, 126, 110, 83, 146, 135, 96, and 36 (SEQ ID NO: 1 or thecorresponding amino acids of SEQ ID NOs: 2-7).

In another embodiment, there is a non-naturally occurring amino acid atposition 91 and position 131 (SEQ ID NO: 1 or the corresponding aminoacids in SEQ ID NOs: 2-7) and one or more of these positions is linkedto a water soluble polymer. In another embodiment, there is anon-naturally occurring amino acid at position 91 and position 77 (SEQID NO: 1 or the corresponding amino acids in SEQ ID NOs: 2-7) and one ormore of these positions is linked to a water soluble polymer. In anotherembodiment, there is a non-naturally occurring amino acid at position 91and position 108 (SEQ ID NO: 1 or the corresponding amino acids in SEQID NOs: 2-7) and one or more of these positions is linked to a watersoluble polymer. In another embodiment, there is a non-naturallyoccurring amino acid at position 131 and position 108 (SEQ ID NO: 1 orthe corresponding amino acids in SEQ ID NOs: 2-7) and one or more ofthese positions is linked to a water soluble polymer. In anotherembodiment, there is a non-naturally occurring amino acid at position131 and position 77 (SEQ ID NO: 1 or the corresponding amino acids inSEQ ID NOs: 2-7) and one or more of these positions is linked to a watersoluble polymer. In another embodiment, there is a non-naturallyoccurring amino acid at position 91 (SEQ ID NO: 1 or the correspondingamino acids in SEQ ID NOs: 2-7) linked to a water soluble polymer. Inanother embodiment, there is a non-naturally occurring amino acid atposition 131 (SEQ ID NO: 1 or the corresponding amino acids in SEQ IDNOs: 2-7) linked to a water soluble polymer. In another embodiment,there is a non-naturally occurring amino acid at position 108 (SEQ IDNO: 1 or the corresponding amino acids in SEQ ID NOs: 2-7) linked to awater soluble polymer. In another embodiment, there is a non-naturallyoccurring amino acid at position 77 (SEQ ID NO: 1 or the correspondingamino acids in SEQ ID NOs: 2-7) linked to a water soluble polymer. Inanother embodiment, there is a non-naturally occurring amino acid atposition 72 (SEQ ID NO: 1 or the corresponding amino acids in SEQ IDNOs: 2-7) linked to a water soluble polymer. In another embodiment,there is a non-naturally occurring amino acid at position 87 (SEQ ID NO:1 or the corresponding amino acids in SEQ ID NOs: 2-7) linked to a watersoluble polymer. In another embodiment, there is a non-naturallyoccurring amino acid at position 86 (SEQ ID NO: 1 or the correspondingamino acids in SEQ ID NOs: 2-7) linked to a water soluble polymer. Inanother embodiment, there is a non-naturally occurring amino acid atposition 126 (SEQ ID NO: 1 or the corresponding amino acids in SEQ IDNOs: 2-7) linked to a water soluble polymer. In another embodiment,there is a non-naturally occurring amino acid at position 110 (SEQ IDNO: 1 or the corresponding amino acids in SEQ ID NOs: 2-7) linked to awater soluble polymer. In another embodiment, there is a non-naturallyoccurring amino acid at position 83 (SEQ ID NO: 1 or the correspondingamino acids in SEQ ID NOs: 2-7) linked to a water soluble polymer.

A wide variety of non-naturally encoded amino acids can be substitutedfor, or incorporated into, a given position in a FGF-21 polypeptide. Ingeneral, a particular non-naturally encoded amino acid is selected forincorporation based on an examination of the three dimensional crystalstructure of a FGF-21 polypeptide or other FGF family member with itsreceptor, a preference for conservative substitutions (i.e., aryl-basednon-naturally encoded amino acids, such as p-acetylphenylalanine orO-propargyltyrosine substituting for Phe, Tyr or Trp), and the specificconjugation chemistry that one desires to introduce into the FGF-21polypeptide (e.g., the introduction of 4-azidophenylalanine if one wantsto effect a Huisgen [3+2] cycloaddition with a water soluble polymerbearing an alkyne moiety or a amide bond formation with a water solublepolymer that bears an aryl ester that, in turn, incorporates a phosphinemoiety).

In one embodiment, the method further includes incorporating into theprotein the unnatural amino acid, where the unnatural amino acidcomprises a first reactive group; and contacting the protein with amolecule (including but not limited to, a label, a dye, a polymer, awater-soluble polymer, a derivative of polyethylene glycol, aphotocrosslinker, a radionuclide, a cytotoxic compound, a drug, anaffinity label, a photoaffinity label, a reactive compound, a resin, asecond protein or polypeptide or polypeptide analog, an antibody orantibody fragment, a metal chelator, a cofactor, a fatty acid, acarbohydrate, a polynucleotide, a DNA, a RNA, an antisensepolynucleotide, a saccharide, a water-soluble dendrimer, a cyclodextrin,an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spinlabel, a fluorophore, a metal-containing moiety, a radioactive moiety, anovel functional group, a group that covalently or noncovalentlyinteracts with other molecules, a photocaged moiety, an actinicradiation excitable moiety, a photoisomerizable moiety, biotin, aderivative of biotin, a biotin analogue, a moiety incorporating a heavyatom, a chemically cleavable group, a photocleavable group, an elongatedside chain, a carbon-linked sugar, a redox-active agent, an aminothioacid, a toxic moiety, an isotopically labeled moiety, a biophysicalprobe, a phosphorescent group, a chemiluminescent group, an electrondense group, a magnetic group, an intercalating group, a chromophore, anenergy transfer agent, a biologically active agent, a detectable label,a small molecule, a quantum dot, a nanotransmitter, a radionucleotide, aradiotransmitter, a neutron-capture agent, or any combination of theabove, or any other desirable compound or substance) that comprises asecond reactive group. The first reactive group reacts with the secondreactive group to attach the molecule to the unnatural amino acidthrough a [3+2] cycloaddition. In one embodiment, the first reactivegroup is an alkynyl or azido moiety and the second reactive group is anazido or alkynyl moiety. For example, the first reactive group is thealkynyl moiety (including but not limited to, in unnatural amino acidp-propargyloxyphenylalanine) and the second reactive group is the azidomoiety. In another example, the first reactive group is the azido moiety(including but not limited to, in the unnatural amino acidp-azido-L-phenylalanine) and the second reactive group is the alkynylmoiety.

In some cases, the non-naturally encoded amino acid substitution(s) willbe combined with other additions, substitutions or deletions within theFGF-21 polypeptide to affect other biological traits of the FGF-21polypeptide. In some cases, the other additions, substitutions ordeletions may increase the stability (including but not limited to,resistance to proteolytic degradation) of the FGF-21 polypeptide orincrease affinity of the FGF-21 polypeptide for its receptor. In somecases, the other additions, substitutions or deletions may increase thepharmaceutical stability of the FGF-21 polypeptide. In some cases, theother additions, substitutions or deletions may increase the solubility(including but not limited to, when expressed in E. coli or other hostcells) of the FGF-21 polypeptide. In some embodiments additions,substitutions or deletions may increase the polypeptide solubilityfollowing expression in E. coli or other recombinant host cells. In someembodiments sites are selected for substitution with a naturally encodedor non-natural amino acid in addition to another site for incorporationof a non-natural amino acid that results in increasing the polypeptidesolubility following expression in E. coli or other recombinant hostcells. In some embodiments, the FGF-21 polypeptides comprise anotheraddition, substitution or deletion that modulates affinity for theFGF-21 polypeptide receptor, binding proteins, or associated ligand,modulates signal transduction after binding to the FGF-21 receptor,modulates circulating half-life, modulates release or bio-availability,facilitates purification, or improves or alters a particular route ofadministration. In some embodiments, the FGF-21 polypeptides comprise anaddition, substitution or deletion that increases the affinity of theFGF-21 variant for its receptor. Similarly, FGF-21 polypeptides cancomprise chemical or enzyme cleavage sequences, protease cleavagesequences, reactive groups, antibody-binding domains (including but notlimited to, FLAG or poly-His) or other affinity based sequences(including, but not limited to, FLAG, poly-His, GST, etc.) or linkedmolecules (including, but not limited to, biotin) that improve detection(including, but not limited to, GFP), purification, transport throughtissues or cell membranes, prodrug release or activation, FGF-21 sizereduction, or other traits of the polypeptide.

In some embodiments, the substitution of a non-naturally encoded aminoacid generates an FGF-21 antagonist. In some embodiments, anon-naturally encoded amino acid is substituted or added in a regioninvolved with receptor binding. In some embodiments, a non-naturallyencoded amino acid is substituted or added in a region involved withheparin binding. In some embodiments, FGF-21 antagonists comprise atleast one substitution that cause FGF-21 to act as an antagonist. Insome embodiments, the FGF-21 antagonist comprises a non-naturallyencoded amino acid linked to a water soluble polymer that is present ina receptor binding region of the FGF-21 molecule.

In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids aresubstituted with one or more non-naturally-encoded amino acids. In somecases, the FGF-21 polypeptide further includes 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more substitutions of one or more non-naturally encoded aminoacids for naturally-occurring amino acids. For example, in someembodiments, one or more residues in FGF-21 are substituted with one ormore non-naturally encoded amino acids. In some cases, the one or morenon-naturally encoded residues are linked to one or more lower molecularweight linear or branched PEGs, thereby enhancing binding affinity andcomparable serum half-life relative to the species attached to a single,higher molecular weight PEG.

In some embodiments, up to two of the following residues of FGF-21 aresubstituted with one or more non-naturally-encoded amino acids.

VII. Expression in Non-Eukaryotes and Eukaryotes

To obtain high level expression of a cloned FGF-21 polynucleotide, onetypically subclones polynucleotides encoding a FGF-21 polypeptide of theinvention into an expression vector that contains a strong promoter todirect transcription, a transcription/translation terminator, and if fora nucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are known tothose of ordinary skill in the art and described, e.g., in Sambrook etal. and Ausubel et al.

Bacterial expression systems for expressing FGF-21 polypeptides of theinvention are available in, including but not limited to, E. coli,Bacillus sp., Pseudomonas fluorescens, Pseudomonas aeruginosa,Pseudomonas putida, and Salmonella (Palva et al., Gene 22:229-235(1983); Mosbach et al., Nature 302:543-545 (1983)). Kits for suchexpression systems are commercially available. Eukaryotic expressionsystems for mammalian cells, yeast, and insect cells are known to thoseof ordinary skill in the art and are also commercially available. Incases where orthogonal tRNAs and aminoacyl tRNA synthetases (describedabove) are used to express the FGF-21 polypeptides of the invention,host cells for expression are selected based on their ability to use theorthogonal components. Exemplary host cells include Gram-positivebacteria (including but not limited to B. brevis, B. subtilis, orStreptomyces) and Gram-negative bacteria (E. coli, Pseudomonasfluorescens, Pseudomonas aeruginosa, Pseudomonas putida), as well asyeast and other eukaryotic cells. Cells comprising O-tRNA/O—RS pairs canbe used as described herein.

A eukaryotic host cell or non-eukaryotic host cell of the presentinvention provides the ability to synthesize proteins that compriseunnatural amino acids in large useful quantities. In one aspect, thecomposition optionally includes, including but not limited to, at least10 micrograms, at least 50 micrograms, at least 75 micrograms, at least100 micrograms, at least 200 micrograms, at least 250 micrograms, atleast 500 micrograms, at least 1 milligram, at least 10 milligrams, atleast 100 milligrams, at least one gram, or more of the protein thatcomprises an unnatural amino acid, or an amount that can be achievedwith in vivo protein production methods (details on recombinant proteinproduction and purification are provided herein). In another aspect, theprotein is optionally present in the composition at a concentration of,including but not limited to, at least 10 micrograms of protein perliter, at least 50 micrograms of protein per liter, at least 75micrograms of protein per liter, at least 100 micrograms of protein perliter, at least 200 micrograms of protein per liter, at least 250micrograms of protein per liter, at least 500 micrograms of protein perliter, at least 1 milligram of protein per liter, or at least 10milligrams of protein per liter or more, in, including but not limitedto, a cell lysate, a buffer, a pharmaceutical buffer, or other liquidsuspension (including but not limited to, in a volume of, including butnot limited to, anywhere from about 1 nl to about 100 L or more). Theproduction of large quantities (including but not limited to, greaterthat that typically possible with other methods, including but notlimited to, in vitro translation) of a protein in a eukaryotic cellincluding at least one unnatural amino acid is a feature of theinvention.

A eukaryotic host cell or non-eukaryotic host cell of the presentinvention provides the ability to biosynthesize proteins that compriseunnatural amino acids in large useful quantities. For example, proteinscomprising an unnatural amino acid can be produced at a concentrationof, including but not limited to, at least 10 μg/liter, at least 50μg/liter, at least 75 μg/liter, at least 100 μg/liter, at least 200μg/liter, at least 250 μg/liter, or at least 500 μg/liter, at least 1mg/liter, at least 2 mg/liter, at least 3 mg/liter, at least 4 mg/liter,at least 5 mg/liter, at least 6 mg/liter, at least 7 mg/liter, at least8 mg/liter, at least 9 mg/liter, at least 10 mg/liter, at least 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900mg/liter, 1 g/liter, 5 g/liter, 10 g/liter or more of protein in a cellextract, cell lysate, culture medium, a buffer, and/or the like.

I. Expression Systems, Culture, and Isolation

FGF-21 polypeptides may be expressed in any number of suitableexpression systems including, for example, yeast, insect cells,mammalian cells, and bacteria. A description of exemplary expressionsystems is provided below.

Yeast As used herein, the term “yeast” includes any of the variousyeasts capable of expressing a gene encoding a FGF-21 polypeptide. Suchyeasts include, but are not limited to, ascosporogenous yeasts(Endomycetales), basidiosporogenous yeasts and yeasts belonging to theFungi imperfecti (Blastomycetes) group. The ascosporogenous yeasts aredivided into two families, Spermophthoraceae and Saccharomycetaceae. Thelatter is comprised of four subfamilies, Schizosaccharomycoideae (e.g.,genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae andSaccharomycoideae (e.g., genera Pichia, Kluyveromyces andSaccharomyces). The basidiosporogenous yeasts include the generaLeucosporidium, Rhodosporidium, Sporidiobolus, Filobasidium, andFilobasidiella. Yeasts belonging to the Fungi Imperfecti (Blastomycetes)group are divided into two families, Sporobolomycetaceae (e.g., generaSporobolomyces and Bullera) and Cryptococcaceae (e.g., genus Candida).

Of particular interest for use with the present invention are specieswithin the genera Pichia, Kluyveromyces, Saccharomyces,Schizosaccharomyces, Hansenula, Torulopsis, and Candida, including, butnot limited to, P. pastoris, P. guillerimondii, S. cerevisiae, S.carlsbergensis, S. diastaticus, S. douglasii, S. kluyveri, S, norbensis,S. oviformis, K lactis, K fragilis, C. albicans, C. maltosa, and H.polymorpha.

The selection of suitable yeast for expression of FGF-21 polypeptides iswithin the skill of one of ordinary skill in the art. In selecting yeasthosts for expression, suitable hosts may include those shown to have,for example, good secretion capacity, low proteolytic activity, goodsecretion capacity, good soluble protein production, and overallrobustness. Yeast are generally available from a variety of sourcesincluding, but not limited to, the Yeast Genetic Stock Center,Department of Biophysics and Medical Physics, University of California(Berkeley, Calif.), and the American Type Culture Collection (“ATCC”)(Manassas, Va.).

The term “yeast host” or “yeast host cell” includes yeast that can be,or has been, used as a recipient for recombinant vectors or othertransfer DNA. The term includes the progeny of the original yeast hostcell that has received the recombinant vectors or other transfer DNA. Itis understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement to the original parent, due to accidental or deliberatemutation. Progeny of the parental cell that are sufficiently similar tothe parent to be characterized by the relevant property, such as thepresence of a nucleotide sequence encoding a FGF-21 polypeptide, areincluded in the progeny intended by this definition.

Expression and transformation vectors, including extrachromosomalreplicons or integrating vectors, have been developed for transformationinto many yeast hosts. For example, expression vectors have beendeveloped for S. cerevisiae (Sikorski et al., GENETICS (1989) 122:19;Ito et al., J. BACTERIOL. (1983) 153:163; Hinnen et al., PROC. NATL.ACAD. SCI. USA (1978) 75:1929); C. albicans (Kurtz et al., MOL. CELL.BIOL. (1986) 6:142); C. maltosa (Kunze et al., J. BASIC MICROBIOL.(1985) 25:141); H. polymorpha (Gleeson et al., J. GEN. MICROBIOL. (1986)132:3459; Roggenkamp et al., MOL. GENETICS AND GENOMICS (1986) 202:302);K. fragilis (Das et al., J. BACTERIOL. (1984) 158:1165); K. lactis (DeLouvencourt et al., J. BACTERIOL. (1983) 154:737; Van den Berg et al.,BIOTECHNOLOGY (NY) (1990) 8:135); P. guillerimondii (Kunze et al., J.BASIC MICROBIOL. (1985) 25:141); P. pastoris (U.S. Pat. Nos. 5,324,639;4,929,555; and 4,837,148; Cregg et al., MOL. CELL. BIOL. (1985) 5:3376);Schizosaccharomyces pombe (Beach et al., NATURE (1982) 300:706); and Y.lipolytica; A. nidulans (Ballance et al., BIOCHEM. BIOPHYS. RES. COMMUN.(1983) 112:284-89; Tilburn et al., GENE (1983) 26:205-221; and Yelton etal., PROC. NATL. ACAD. SCI. USA (1984) 81:1470-74); A. niger (Kelly andHynes, EMBO J. (1985) 4:475-479); T reesia (EP 0 244 234); andfilamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium(WO 91/00357), each incorporated by reference herein.

Control sequences for yeast vectors are known to those of ordinary skillin the art and include, but are not limited to, promoter regions fromgenes such as alcohol dehydrogenase (ADH) (EP 0 284 044); enolase;glucokinase; glucose-6-phosphate isomerase;glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH); hexokinase;phosphofructokinase; 3-phosphoglycerate mutase; and pyruvate kinase(PyK) (EP 0 329 203). The yeast PHOS gene, encoding acid phosphatase,also may provide useful promoter sequences (Miyanohara et al., PROC.NATL. ACAD. SCI. USA (1983) 80:1). Other suitable promoter sequences foruse with yeast hosts may include the promoters for 3-phosphoglyceratekinase (Hitzeman et al., J. BIOL. CHEM. (1980) 255:12073); and otherglycolytic enzymes, such as pyruvate decarboxylase, triosephosphateisomerase, and phosphoglucose isomerase (Holland et al., BIOCHEMISTRY(1978) 17:4900; Hess et al., J. ADV. ENZYME REG. (1969) 7:149).Inducible yeast promoters having the additional advantage oftranscription controlled by growth conditions may include the promoterregions for alcohol dehydrogenase 2; isocytochrome C; acid phosphatase;metallothionein; glyceraldehyde-3-phosphate dehydrogenase; degradativeenzymes associated with nitrogen metabolism; and enzymes responsible formaltose and galactose utilization. Suitable vectors and promoters foruse in yeast expression are further described in EP 0 073 657.

Yeast enhancers also may be used with yeast promoters. In addition,synthetic promoters may also function as yeast promoters. For example,the upstream activating sequences (UAS) of a yeast promoter may bejoined with the transcription activation region of another yeastpromoter, creating a synthetic hybrid promoter. Examples of such hybridpromoters include the ADH regulatory sequence linked to the GAPtranscription activation region. See U.S. Pat. Nos. 4,880,734 and4,876,197, which are incorporated by reference herein. Other examples ofhybrid promoters include promoters that consist of the regulatorysequences of the ADH2, GAL4, GAL10, or PHOS genes, combined with thetranscriptional activation region of a glycolytic enzyme gene such asGAP or PyK. See EP 0 164 556. Furthermore, a yeast promoter may includenaturally occurring promoters of non-yeast origin that have the abilityto bind yeast RNA polymerase and initiate transcription.

Other control elements that may comprise part of the yeast expressionvectors include terminators, for example, from GAPDH or the enolasegenes (Holland et al., J. BIOL. CHEM. (1981) 256:1385). In addition, theorigin of replication from the 2μ plasmid origin is suitable for yeast.A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid. See Tschumper et al., GENE (1980) 10:157; Kingsman etal., GENE (1979) 7:141. The trp1 gene provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan.Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) arecomplemented by known plasmids bearing the Leu2 gene.

Methods of introducing exogenous DNA into yeast hosts are known to thoseof ordinary skill in the art, and typically include, but are not limitedto, either the transformation of spheroplasts or of intact yeast hostcells treated with alkali cations. For example, transformation of yeastcan be carried out according to the method described in Hsiao et al.,PROC. NATL. ACAD. SCI. USA (1979) 76:3829 and Van Solingen et al., J.BACT. (1977) 130:946. However, other methods for introducing DNA intocells such as by nuclear injection, electroporation, or protoplastfusion may also be used as described generally in SAMBROOK ET AL.,MOLECULAR CLONING: A LAB. MANUAL (2001). Yeast host cells may then becultured using standard techniques known to those of ordinary skill inthe art.

Other methods for expressing heterologous proteins in yeast host cellsare known to those of ordinary skill in the art. See generally U.S.Patent Publication No. 20020055169, U.S. Pat. Nos. 6,361,969; 6,312,923;6,183,985; 6,083,723; 6,017,731; 5,674,706; 5,629,203; 5,602,034; and5,089,398; U.S. Reexamined Patent Nos. RE37,343 and RE35,749; PCTPublished Patent Applications WO 99/07862; WO 98/37208; and WO 98/26080;European Patent Applications EP 0 946 736; EP 0 732 403; EP 0 480 480;WO 90/10277; EP 0 340 986; EP 0 329 203; EP 0 324 274; and EP 0 164 556.See also Gellissen et al., ANTONIE VAN LEEUWENHOEK (1992) 62(1-2):79-93;Romanos et al., YEAST (1992) 8(6):423-488; Goeddel, METHODS INENZYMOLOGY (1990) 185:3-7, each incorporated by reference herein.

The yeast host strains may be grown in fermentors during theamplification stage using standard feed batch fermentation methods knownto those of ordinary skill in the art. The fermentation methods may beadapted to account for differences in a particular yeast host's carbonutilization pathway or mode of expression control. For example,fermentation of a Saccharomyces yeast host may require a single glucosefeed, complex nitrogen source (e.g., casein hydrolysates), and multiplevitamin supplementation. In contrast, the methylotrophic yeast P.pastoris may require glycerol, methanol, and trace mineral feeds, butonly simple ammonium (nitrogen) salts for optimal growth and expression.See, e.g., U.S. Pat. No. 5,324,639; Elliott et al., J. PROTEIN CHEM.(1990) 9:95; and Fieschko et al., BIOTECH. BIOENG. (1987) 29:1113,incorporated by reference herein.

Such fermentation methods, however, may have certain common featuresindependent of the yeast host strain employed. For example, a growthlimiting nutrient, typically carbon, may be added to the fermentorduring the amplification phase to allow maximal growth. In addition,fermentation methods generally employ a fermentation medium designed tocontain adequate amounts of carbon, nitrogen, basal salts, phosphorus,and other minor nutrients (vitamins, trace minerals and salts, etc.).Examples of fermentation media suitable for use with Pichia aredescribed in U.S. Pat. Nos. 5,324,639 and 5,231,178, which areincorporated by reference herein. WO 2005/091944 which is incorporatedby reference herein describes the expression of FGF-21 in yeast.

Baculovirus-Infected Insect Cells The term “insect host” or “insect hostcell” refers to a insect that can be, or has been, used as a recipientfor recombinant vectors or other transfer DNA. The term includes theprogeny of the original insect host cell that has been transfected. Itis understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement to the original parent, due to accidental or deliberatemutation. Progeny of the parental cell that are sufficiently similar tothe parent to be characterized by the relevant property, such as thepresence of a nucleotide sequence encoding a FGF-21 polypeptide, areincluded in the progeny intended by this definition.

The selection of suitable insect cells for expression of FGF-21polypeptides is known to those of ordinary skill in the art. Severalinsect species are well described in the art and are commerciallyavailable including Aedes aegypti, Bombyx mori, Drosophila melanogaster,Spodoptera frupperda, and Trichoplusia ni. In selecting insect hosts forexpression, suitable hosts may include those shown to have, inter alia,good secretion capacity, low proteolytic activity, and overallrobustness. Insect are generally available from a variety of sourcesincluding, but not limited to, the Insect Genetic Stock Center,Department of Biophysics and Medical Physics, University of California(Berkeley, Calif.); and the American Type Culture Collection (“ATCC”)(Manassas, Va.).

Generally, the components of a baculovirus-infected insect expressionsystem include a transfer vector, usually a bacterial plasmid, whichcontains both a fragment of the baculovirus genome, and a convenientrestriction site for insertion of the heterologous gene to be expressed;a wild type baculovirus with sequences homologous to thebaculovirus-specific fragment in the transfer vector (this allows forthe homologous recombination of the heterologous gene in to thebaculovirus genome); and appropriate insect host cells and growth media.The materials, methods and techniques used in constructing vectors,transfecting cells, picking plaques, growing cells in culture, and thelike are known in the art and manuals are available describing thesetechniques.

After inserting the heterologous gene into the transfer vector, thevector and the wild type viral genome are transfected into an insecthost cell where the vector and viral genome recombine. The packagedrecombinant virus is expressed and recombinant plaques are identifiedand purified. Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, forexample, Invitrogen Corp. (Carlsbad, Calif.). These techniques aregenerally known to those of ordinary skill in the art and fullydescribed in SUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENT STATIONBULLETIN NO. 1555 (1987), herein incorporated by reference. See also,RICHARDSON, 39 METHODS IN MOLECULAR BIOLOGY: BACULOVIRUS EXPRESSIONPROTOCOLS (1995); AUSUBEL ET AL., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY16.9-16.11 (1994); KING AND POSSEE, THE BACULOVIRUS SYSTEM: A LABORATORYGUIDE (1992); and O'REILLY ET AL., BACULOVIRUS EXPRESSION VECTORS: ALABORATORY MANUAL (1992).

Indeed, the production of various heterologous proteins usingbaculovirus/insect cell expression systems is known to those of ordinaryskill in the art. See, e.g., U.S. Pat. Nos. 6,368,825; 6,342,216;6,338,846; 6,261,805; 6,245,528, 6,225,060; 6,183,987; 6,168,932;6,126,944; 6,096,304; 6,013,433; 5,965,393; 5,939,285; 5,891,676;5,871,986; 5,861,279; 5,858,368; 5,843,733; 5,762,939; 5,753,220;5,605,827; 5,583,023; 5,571,709; 5,516,657; 5,290,686; WO 02/06305; WO01/90390; WO 01/27301; WO 01/05956; WO 00/55345; WO 00/20032; WO99/51721; WO 99/45130; WO 99/31257; WO 99/10515; WO 99/09193; WO97/26332; WO 96/29400; WO 96/25496; WO 96/06161; WO 95/20672; WO93/03173; WO 92/16619; WO 92/02628; WO 92/01801; WO 90/14428; WO90/10078; WO 90/02566; WO 90/02186; WO 90/01556; WO 89/01038; WO89/01037; WO 88/07082, which are incorporated by reference herein.

Vectors that are useful in baculovirus/insect cell expression systemsare known in the art and include, for example, insect expression andtransfer vectors derived from the baculovirus Autographacalifornicanuclear polyhedrosis virus (AcNPV), which is a helper-independent, viralexpression vector. Viral expression vectors derived from this systemusually use the strong viral polyhedrin gene promoter to driveexpression of heterologous genes. See generally, O'Reilly ET AL.,BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992).

Prior to inserting the foreign gene into the baculovirus genome, theabove-described components, comprising a promoter, leader (if desired),coding sequence of interest, and transcription termination sequence, aretypically assembled into an intermediate transplacement construct(transfer vector). Intermediate transplacement constructs are oftenmaintained in a replicon, such as an extra chromosomal element (e.g.,plasmids) capable of stable maintenance in a host, such as bacteria. Thereplicon will have a replication system, thus allowing it to bemaintained in a suitable host for cloning and amplification. Morespecifically, the plasmid may contain the polyhedrin polyadenylationsignal (Miller, ANN. REV. MICROBIOL. (1988) 42:177) and a prokaryoticampicillin-resistance (amp) gene and origin of replication for selectionand propagation in E. coli.

One commonly used transfer vector for introducing foreign genes intoAcNPV is pAc373. Many other vectors, known to those of skill in the art,have also been designed including, for example, pVL985, which alters thepolyhedrin start codon from ATG to ATT, and which introduces a BamHIcloning site 32 base pairs downstream from the ATT. See Luckow andSummers, VIROLOGY 170:31 (1989). Other commercially available vectorsinclude, for example, PBlueBac4.5/V5-His; pBlueBacHis2; pMelBac;pBlueBac4.5 (Invitrogen Corp., Carlsbad, Calif.).

After insertion of the heterologous gene, the transfer vector and wildtype baculoviral genome are co-transfected into an insect cell host.Methods for introducing heterologous DNA into the desired site in thebaculovirus virus are known in the art. See SUMMERS AND SMITH, TEXASAGRICULTURAL EXPERIMENT STATION BULLETIN NO. 1555 (1987); Smith et al.,MOL. CELL. BIOL. (1983) 3:2156; Luckow and Summers, VIROLOGY (1989)170:31. For example, the insertion can be into a gene such as thepolyhedrin gene, by homologous double crossover recombination; insertioncan also be into a restriction enzyme site engineered into the desiredbaculovirus gene. See Miller et al., BIOESSAYS (1989) 11(4):91.

Transfection may be accomplished by electroporation. See TROTTER ANDWOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Mann and King, J. GEN.VIROL. (1989) 70:3501. Alternatively, liposomes may be used to transfectthe insect cells with the recombinant expression vector and thebaculovirus. See, e.g., Liebman et al., BIOTECHNIQUES (1999) 26(1):36;Graves et al., BIOCHEMISTRY (1998) 37:6050; Nomura et al., J. BIOL.CHEM. (1998) 273(22):13570; Schmidt et al., PROTEIN EXPRESSION ANDPURIFICATION (1998) 12:323; Siffert et al., NATURE GENETICS (1998)18:45; TILKINS ET AL., CELL BIOLOGY: A LABORATORY HANDBOOK 145-154(1998); Cai et al., PROTEIN EXPRESSION AND PURIFICATION (1997) 10:263;Dolphin et al., NATURE GENETICS (1997) 17:491; Kost et al., GENE (1997)190:139; Jakobsson et al., J. BIOL. CHEM. (1996) 271:22203; Rowles etal., J. BIOL. CHEM. (1996) 271(37):22376; Reverey et al., J. BIOL. CHEM.(1996) 271(39):23607-10; Stanley et al., J. BIOL. CHEM. (1995) 270:4121;Sisk et al., J. VIROL. (1994) 68(2):766; and Peng et al., BIOTECHNIQUES(1993) 14(2):274. Commercially available liposomes include, for example,Cellfectin® and Lipofectin® (Invitrogen, Corp., Carlsbad, Calif.). Inaddition, calcium phosphate transfection may be used. See TROTTER ANDWOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Kitts, NAR (1990)18(19):5667; and Mann and King, J. GEN. VIROL. (1989) 70:3501.

Baculovirus expression vectors usually contain a baculovirus promoter. Abaculovirus promoter is any DNA sequence capable of binding abaculovirus RNA polymerase and initiating the downstream (3′)transcription of a coding sequence (e.g., structural gene) into mRNA. Apromoter will have a transcription initiation region which is usuallyplaced proximal to the 5′ end of the coding sequence. This transcriptioninitiation region typically includes an RNA polymerase binding site anda transcription initiation site. A baculovirus promoter may also have asecond domain called an enhancer, which, if present, is usually distalto the structural gene. Moreover, expression may be either regulated orconstitutive.

Structural genes, abundantly transcribed at late times in the infectioncycle, provide particularly useful promoter sequences. Examples includesequences derived from the gene encoding the viral polyhedron protein(FRIESEN ET AL., The Regulation of Baculovirus Gene Expression in THEMOLECULAR BIOLOGY OF BACULOVIRUSES (1986); EP 0 127 839 and 0 155 476)and the gene encoding the p10 protein (Vlak et al., J. GEN. VIROL.(1988) 69:765).

The newly formed baculovirus expression vector is packaged into aninfectious recombinant baculovirus and subsequently grown plaques may bepurified by techniques known to those of ordinary skill in the art. SeeMiller et al., BIOESSAYS (1989) 11(4):91; SUMMERS AND SMITH, TEXASAGRICULTURAL EXPERIMENT STATION BULLETIN NO. 1555 (1987).

Recombinant baculovirus expression vectors have been developed forinfection into several insect cells. For example, recombinantbaculoviruses have been developed for, inter alia, Aedes aegypti (ATCCNo. CCL-125), Bombyx mori (ATCC No. CRL-8910), Drosophila melanogaster(ATCC No. 1963), Spodoptera frupperda, and Trichoplusia ni. See Wright,NATURE (1986) 321:718; Carbonell et al., J. VIROL. (1985) 56:153; Smithet al., MOL. CELL. BIOL. (1983) 3:2156. See generally, Fraser et al., INVITRO CELL. DEV. BIOL. (1989) 25:225. More specifically, the cell linesused for baculovirus expression vector systems commonly include, but arenot limited to, Sf9 (Spodoptera frupperda) (ATCC No. CRL-1711), Sf21(Spodoptera frupperda) (Invitrogen Corp., Cat. No. 11497-013 (Carlsbad,Calif.)), Tri-368 (Trichopulsia ni), and High-Five™ BTI-TN-5B1-4(Trichopulsia ni).

Cells and culture media are commercially available for both direct andfusion expression of heterologous polypeptides in abaculovirus/expression, and cell culture technology is generally knownto those of ordinary skill in the art.

E. Coli, Pseudomonas species, and other Prokaryotes Bacterial expressiontechniques are known to those of ordinary skill in the art. A widevariety of vectors are available for use in bacterial hosts. The vectorsmay be single copy or low or high multicopy vectors. Vectors may servefor cloning and/or expression. In view of the ample literatureconcerning vectors, commercial availability of many vectors, and evenmanuals describing vectors and their restriction maps andcharacteristics, no extensive discussion is required here. As iswell-known, the vectors normally involve markers allowing for selection,which markers may provide for cytotoxic agent resistance, prototrophy orimmunity. Frequently, a plurality of markers is present, which providefor different characteristics.

A bacterial promoter is any DNA sequence capable of binding bacterialRNA polymerase and initiating the downstream (3′) transcription of acoding sequence (e.g. structural gene) into mRNA. A promoter will have atranscription initiation region which is usually placed proximal to the5′ end of the coding sequence. This transcription initiation regiontypically includes an RNA polymerase binding site and a transcriptioninitiation site. A bacterial promoter may also have a second domaincalled an operator, that may overlap an adjacent RNA polymerase bindingsite at which RNA synthesis begins. The operator permits negativeregulated (inducible) transcription, as a gene repressor protein maybind the operator and thereby inhibit transcription of a specific gene.Constitutive expression may occur in the absence of negative regulatoryelements, such as the operator. In addition, positive regulation may beachieved by a gene activator protein binding sequence, which, if presentis usually proximal (5′) to the RNA polymerase binding sequence. Anexample of a gene activator protein is the catabolite activator protein(CAP), which helps initiate transcription of the lac operon inEscherichia coli (E. coli) [Raibaud et al., ANNU. REV. GENET. (1984)18:173]. Regulated expression may therefore be either positive ornegative, thereby either enhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose (lac) [Chang etal., NATURE (1977) 198:1056], and maltose. Additional examples includepromoter sequences derived from biosynthetic enzymes such as tryptophan(trp) [Goeddel et al., NUC. ACIDS RES. (1980) 8:4057; Yelverton et al.,NUCL. ACIDS RES. (1981) 9:731; U.S. Pat. No. 4,738,921; EP Pub. Nos. 036776 and 121 775, which are incorporated by reference herein]. Theβ-galactosidase (bla) promoter system [Weissmann (1981) “The cloning ofinterferon and other mistakes.” In Interferon 3 (Ed. I. Gresser)],bacteriophage lambda PL [Shimatake et al., NATURE (1981) 292:128] and T5[U.S. Pat. No. 4,689,406, which are incorporated by reference herein]promoter systems also provide useful promoter sequences. Preferredmethods of the present invention utilize strong promoters, such as theT7 promoter to induce FGF-21 polypeptides at high levels. Examples ofsuch vectors are known to those of ordinary skill in the art and includethe pET29 series from Novagen, and the pPOP vectors described inWO99/05297, which is incorporated by reference herein. Such expressionsystems produce high levels of FGF-21 polypeptides in the host withoutcompromising host cell viability or growth parameters. pET19 (Novagen)is another vector known in the art.

In addition, synthetic promoters which do not occur in nature alsofunction as bacterial promoters. For example, transcription activationsequences of one bacterial or bacteriophage promoter may be joined withthe operon sequences of another bacterial or bacteriophage promoter,creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433, which isincorporated by reference herein]. For example, the tac promoter is ahybrid trp-lac promoter comprised of both trp promoter and lac operonsequences that is regulated by the lac repressor [Amann et al., GENE(1983) 25:167; de Boer et al., PROC. NATL. ACAD. SCI. (1983) 80:21].Furthermore, a bacterial promoter can include naturally occurringpromoters of non-bacterial origin that have the ability to bindbacterial RNA polymerase and initiate transcription. A naturallyoccurring promoter of non-bacterial origin can also be coupled with acompatible RNA polymerase to produce high levels of expression of somegenes in prokaryotes. The bacteriophage T7 RNA polymerase/promotersystem is an example of a coupled promoter system [Studier et al., J.MOL. BIOL. (1986) 189:113; Tabor et al., Proc Natl. Acad. Sci. (1985)82:1074]. In addition, a hybrid promoter can also be comprised of abacteriophage promoter and an E. coli operator region (EP Pub. No. 267851).

In addition to a functioning promoter sequence, an efficient ribosomebinding site is also useful for the expression of foreign genes inprokaryotes. In E. coli, the ribosome binding site is called theShine-Dalgarno (SD) sequence and includes an initiation codon (ATG) anda sequence 3-9 nucleotides in length located 3-11 nucleotides upstreamof the initiation codon [Shine et al., NATURE (1975) 254:34]. The SDsequence is thought to promote binding of mRNA to the ribosome by thepairing of bases between the SD sequence and the 3′ and of E. coli 16SrRNA [Steitz et al. “Genetic signals and nucleotide sequences inmessenger RNA”, In Biological Regulation and Development: GeneExpression (Ed. R. F. Goldberger, 1979)]. To express eukaryotic genesand prokaryotic genes with weak ribosome-binding site [Sambrook et al.“Expression of cloned genes in Escherichia coli”, Molecular Cloning: ALaboratory Manual, 1989].

The term “bacterial host” or “bacterial host cell” refers to a bacterialthat can be, or has been, used as a recipient for recombinant vectors orother transfer DNA. The term includes the progeny of the originalbacterial host cell that has been transfected. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement to theoriginal parent, due to accidental or deliberate mutation. Progeny ofthe parental cell that are sufficiently similar to the parent to becharacterized by the relevant property, such as the presence of anucleotide sequence encoding a FGF-21 polypeptide, are included in theprogeny intended by this definition.

The selection of suitable host bacteria for expression of FGF-21polypeptides is known to those of ordinary skill in the art. Inselecting bacterial hosts for expression, suitable hosts may includethose shown to have, inter alia, good inclusion body formation capacity,low proteolytic activity, and overall robustness. Bacterial hosts aregenerally available from a variety of sources including, but not limitedto, the Bacterial Genetic Stock Center, Department of Biophysics andMedical Physics, University of California (Berkeley, Calif.); and theAmerican Type Culture Collection (“ATCC”) (Manassas, Va.).Industrial/pharmaceutical fermentation generally use bacterial derivedfrom K strains (e.g. W3110) or from bacteria derived from B strains(e.g. BL21). These strains are particularly useful because their growthparameters are extremely well known and robust. In addition, thesestrains are non-pathogenic, which is commercially important for safetyand environmental reasons. Other examples of suitable E. coli hostsinclude, but are not limited to, strains of BL21, DH10B, or derivativesthereof. In another embodiment of the methods of the present invention,the E. coli host is a protease minus strain including, but not limitedto, OMP- and LON-. The host cell strain may be a species of Pseudomonas,including but not limited to, Pseudomonas fluorescens, Pseudomonasaeruginosa, and Pseudomonas putida. Pseudomonas fluorescens biovar 1,designated strain MB101, is known to be useful for recombinantproduction and is available for therapeutic protein productionprocesses. Examples of a Pseudomonas expression system include thesystem available from The Dow Chemical Company as a host strain(Midland, Mich. available on the World Wide Web at dow.com).

Once a recombinant host cell strain has been established (i.e., theexpression construct has been introduced into the host cell and hostcells with the proper expression construct are isolated), therecombinant host cell strain is cultured under conditions appropriatefor production of FGF-21 polypeptides. As will be apparent to one ofskill in the art, the method of culture of the recombinant host cellstrain will be dependent on the nature of the expression constructutilized and the identity of the host cell. Recombinant host strains arenormally cultured using methods that are known to those of ordinaryskill in the art. Recombinant host cells are typically cultured inliquid medium containing assimilatable sources of carbon, nitrogen, andinorganic salts and, optionally, containing vitamins, amino acids,growth factors, and other proteinaceous culture supplements known tothose of ordinary skill in the art. Liquid media for culture of hostcells may optionally contain antibiotics or anti-fungals to prevent thegrowth of undesirable microorganisms and/or compounds including, but notlimited to, antibiotics to select for host cells containing theexpression vector.

Recombinant host cells may be cultured in batch or continuous formats,with either cell harvesting (in the case where the FGF-21 polypeptideaccumulates intracellularly) or harvesting of culture supernatant ineither batch or continuous formats. For production in prokaryotic hostcells, batch culture and cell harvest are preferred.

The FGF-21 polypeptides of the present invention are normally purifiedafter expression in recombinant systems. The FGF-21 polypeptide may bepurified from host cells or culture medium by a variety of methods knownto the art. FGF-21 polypeptides produced in bacterial host cells may bepoorly soluble or insoluble (in the form of inclusion bodies). In oneembodiment of the present invention, amino acid substitutions mayreadily be made in the FGF-21 polypeptide that are selected for thepurpose of increasing the solubility of the recombinantly producedprotein utilizing the methods disclosed herein as well as those known inthe art. In the case of insoluble protein, the protein may be collectedfrom host cell lysates by centrifugation and may further be followed byhomogenization of the cells. In the case of poorly soluble protein,compounds including, but not limited to, polyethylene imine (PEI) may beadded to induce the precipitation of partially soluble protein. Theprecipitated protein may then be conveniently collected bycentrifugation. Recombinant host cells may be disrupted or homogenizedto release the inclusion bodies from within the cells using a variety ofmethods known to those of ordinary skill in the art. Host celldisruption or homogenization may be performed using well knowntechniques including, but not limited to, enzymatic cell disruption,sonication, dounce homogenization, or high pressure release disruption.In one embodiment of the method of the present invention, the highpressure release technique is used to disrupt the E. coli host cells torelease the inclusion bodies of the FGF-21 polypeptides. When handlinginclusion bodies of FGF-21 polypeptide, it may be advantageous tominimize the homogenization time on repetitions in order to maximize theyield of inclusion bodies without loss due to factors such assolubilization, mechanical shearing or proteolysis.

Insoluble or precipitated FGF-21 polypeptide may then be solubilizedusing any of a number of suitable solubilization agents known to theart. The FGF-21 polyeptide may be solubilized with urea or guanidinehydrochloride. The volume of the solubilized FGF-21 polypeptide shouldbe minimized so that large batches may be produced using convenientlymanageable batch sizes. This factor may be significant in a large-scalecommercial setting where the recombinant host may be grown in batchesthat are thousands of liters in volume. In addition, when manufacturingFGF-21 polypeptide in a large-scale commercial setting, in particularfor human pharmaceutical uses, the avoidance of harsh chemicals that candamage the machinery and container, or the protein product itself,should be avoided, if possible. It has been shown in the method of thepresent invention that the milder denaturing agent urea can be used tosolubilize the FGF-21 polypeptide inclusion bodies in place of theharsher denaturing agent guanidine hydrochloride. The use of ureasignificantly reduces the risk of damage to stainless steel equipmentutilized in the manufacturing and purification process of FGF-21polypeptide while efficiently solubilizing the FGF-21 polypeptideinclusion bodies.

In the case of soluble FGF-21 protein, the FGF-21 may be secreted intothe periplasmic space or into the culture medium. For example, FGF-21was secreted into the periplasmic space of W3110-B2 cells by usingplasmids encoding constructs including eight different leader sequences,including those listed in SEQ ID NOs: 39-44, and transforming these intoW3110-B2 cells, the cells were then grown at 37° C. until OD reachedabout 0.8, at which point the expression was induced with 0.01%arabinose. Five hours later the periplasmic release samples were preppedfrom the cultures and run on the gels (FIG. 33) showing overallexpression (total lysates) and periplasmic secretions (solublefraction).

In addition, soluble FGF-21 may be present in the cytoplasm of the hostcells. It may be desired to concentrate soluble FGF-21 prior toperforming purification steps. Standard techniques known to those ofordinary skill in the art may be used to concentrate soluble FGF-21from, for example, cell lysates or culture medium. In addition, standardtechniques known to those of ordinary skill in the art may be used todisrupt host cells and release soluble FGF-21 from the cytoplasm orperiplasmic space of the host cells.

When FGF-21 polypeptide is produced as a fusion protein, the fusionsequence may be removed. Removal of a fusion sequence may beaccomplished by enzymatic or chemical cleavage. Enzymatic removal offusion sequences may be accomplished using methods known to those ofordinary skill in the art. The choice of enzyme for removal of thefusion sequence will be determined by the identity of the fusion, andthe reaction conditions will be specified by the choice of enzyme aswill be apparent to one of ordinary skill in the art. Chemical cleavagemay be accomplished using reagents known to those of ordinary skill inthe art, including but not limited to, cyanogen bromide, TEV protease,and other reagents. The cleaved FGF-21 polypeptide may be purified fromthe cleaved fusion sequence by methods known to those of ordinary skillin the art. Such methods will be determined by the identity andproperties of the fusion sequence and the FGF-21 polypeptide, as will beapparent to one of ordinary skill in the art. Methods for purificationmay include, but are not limited to, size-exclusion chromatography,hydrophobic interaction chromatography, ion-exchange chromatography ordialysis or any combination thereof.

The FGF-21 polypeptide may also be purified to remove DNA from theprotein solution. DNA may be removed by any suitable method known to theart, such as precipitation or ion exchange chromatography, but may beremoved by precipitation with a nucleic acid precipitating agent, suchas, but not limited to, protamine sulfate. The FGF-21 polypeptide may beseparated from the precipitated DNA using standard well known methodsincluding, but not limited to, centrifugation or filtration. Removal ofhost nucleic acid molecules is an important factor in a setting wherethe FGF-21 polypeptide is to be used to treat humans and the methods ofthe present invention reduce host cell DNA to pharmaceuticallyacceptable levels.

Methods for small-scale or large-scale fermentation can also be used inprotein expression, including but not limited to, fermentors, shakeflasks, fluidized bed bioreactors, hollow fiber bioreactors, rollerbottle culture systems, and stirred tank bioreactor systems. Each ofthese methods can be performed in a batch, fed-batch, or continuous modeprocess.

Human FGF-21 polypeptides of the invention can generally be recoveredusing methods standard in the art. For example, culture medium or celllysate can be centrifuged or filtered to remove cellular debris. Thesupernatant may be concentrated or diluted to a desired volume ordiafiltered into a suitable buffer to condition the preparation forfurther purification. Further purification of the FGF-21 polypeptide ofthe present invention includes separating deamidated and clipped formsof the FGF-21 polypeptide variant from the intact form.

Any of the following exemplary procedures can be employed forpurification of FGF-polypeptides of the invention: affinitychromatography; anion- or cation-exchange chromatography (using,including but not limited to, DEAE SEPHAROSE); chromatography on silica;high performance liquid chromatography (HPLC); reverse phase HPLC; gelfiltration (using, including but not limited to, SEPHADEX G-75);hydrophobic interaction chromatography; size-exclusion chromatography;metal-chelate chromatography; ultrafiltration/diafiltration; ethanolprecipitation; ammonium sulfate precipitation; chromatofocusing;displacement chromatography; electrophoretic procedures (including butnot limited to preparative isoelectric focusing), differentialsolubility (including but not limited to ammonium sulfateprecipitation), SD S-PAGE, or extraction.

Proteins of the present invention, including but not limited to,proteins comprising unnatural amino acids, peptides comprising unnaturalamino acids, antibodies to proteins comprising unnatural amino acids,binding partners for proteins comprising unnatural amino acids, etc.,can be purified, either partially or substantially to homogeneity,according to standard procedures known to and used by those of skill inthe art. Accordingly, polypeptides of the invention can be recovered andpurified by any of a number of methods known to those of ordinary skillin the art, including but not limited to, ammonium sulfate or ethanolprecipitation, acid or base extraction, column chromatography, affinitycolumn chromatography, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,hydroxylapatite chromatography, lectin chromatography, gelelectrophoresis and the like. Protein refolding steps can be used, asdesired, in making correctly folded mature proteins. High performanceliquid chromatography (HPLC), affinity chromatography or other suitablemethods can be employed in final purification steps where high purity isdesired. In one embodiment, antibodies made against unnatural aminoacids (or proteins or peptides comprising unnatural amino acids) areused as purification reagents, including but not limited to, foraffinity-based purification of proteins or peptides comprising one ormore unnatural amino acid(s). Once purified, partially or tohomogeneity, as desired, the polypeptides are optionally used for a widevariety of utilities, including but not limited to, as assay components,therapeutics, prophylaxis, diagnostics, research reagents, and/or asimmunogens for antibody production. Antibodies generated againstpolypeptides of the present invention may be obtained by administeringthe polypeptides or epitope-bearing fragments, or cells to an animal,preferably a non-human animal, using routine protocols. One of ordinaryskill in the art could generate antibodies using a variety of knowntechniques. Also, transgenic mice, or other organisms, including othermammals, may be used to express humanized antibodies. Theabove-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptides.Antibodies against polypeptides of the present invention may also beemployed to treat diseases.

Polypeptides and polynucleotides of the present invention may also beused as vaccines. Accordingly, in a further aspect, the presentinvention relates to a method for inducing an immunological response ina mammal that comprises inoculating the mammal with a polypeptide of thepresent invention, adequate to produce antibody and/or T cell immuneresponse, including, for example, cytokine-producing T cells orcytotoxic T cells, to protect said animal from disease, whether thatdisease is already established within the individual or not. Animmunological response in a mammal may also be induced by a methodcomprises delivering a polypeptide of the present invention via a vectordirecting expression of the polynucleotide and coding for thepolypeptide in vivo in order to induce such an immunological response toproduce antibody to protect said animal from diseases of the invention.One way of administering the vector is by accelerating it into thedesired cells as a coating on particles or otherwise. Such nucleic acidvector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNAhybrid. For use as a vaccine, a polypeptide or a nucleic acid vectorwill be normally provided as a vaccine formulation (composition). Theformulation may further comprise a suitable carrier. Since a polypeptidemay be broken down in the stomach, it may be administered parenterally(for instance, subcutaneous, intramuscular, intravenous, or intra-dermalinjection). Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions that may containanti-oxidants, buffers, bacteriostats and solutes that render theformulation instonic with the blood of the recipient; and aqueous andnon-aqueous sterile suspensions that may include suspending agents orthickening agents. The vaccine formulation may also include adjuvantsystems for enhancing the immunogenicity of the formulation which areknown to those of ordinary skill in the art. The dosage will depend onthe specific activity of the vaccine and can be readily determined byroutine experimentation.

In addition to other references noted herein, a variety ofpurification/protein folding methods are known to those of ordinaryskill in the art, including, but not limited to, those set forth in R.Scopes, Protein Purification, Springer-Verlag, N.Y. (1982); Deutscher,Methods in Enzymology Vol. 182: Guide to Protein Purification, AcademicPress, Inc. N.Y. (1990); Sandana, (1997) Bioseparation of Proteins,Academic Press, Inc.; Bollag et al. (1996) Protein Methods, 2nd EditionWiley-Liss, NY; Walker, (1996) The Protein Protocols Handbook HumanaPress, NJ, Harris and Angal, (1990) Protein Purification Applications: APractical Approach IRL Press at Oxford, Oxford, England; Harris andAngal, Protein Purification Methods: A Practical Approach IRL Press atOxford, Oxford, England; Scopes, (1993) Protein Purification: Principlesand Practice 3rd Edition Springer Verlag, NY; Janson and Ryden, (1998)Protein Purification: Principles, High Resolution Methods andApplications, Second Edition Wiley-VCH, NY; and Walker (1998), ProteinProtocols on CD-ROM Humana Press, NJ; and the references cited therein.

One advantage of producing a protein or polypeptide of interest with anunnatural amino acid in a eukaryotic host cell or non-eukaryotic hostcell is that typically the proteins or polypeptides will be folded intheir native conformations. However, in certain embodiments of theinvention, those of skill in the art will recognize that, aftersynthesis, expression and/or purification, proteins or peptides canpossess a conformation different from the desired conformations of therelevant polypeptides. In one aspect of the invention, the expressedprotein or polypeptide is optionally denatured and then renatured. Thisis accomplished utilizing methods known in the art, including but notlimited to, by adding a chaperonin to the protein or polypeptide ofinterest, by solubilizing the proteins in a chaotropic agent such asguanidine HCl, utilizing protein disulfide isomerase, etc.

In general, it is occasionally desirable to denature and reduceexpressed polypeptides and then to cause the polypeptides to re-foldinto the preferred conformation. For example, guanidine, urea, DTT, DTE,and/or a chaperonin can be added to a translation product of interest.Methods of reducing, denaturing and renaturing proteins are known tothose of ordinary skill in the art (see, the references above, andDebinski, et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman andPastan (1993) Bioconjug. Chem., 4: 581-585; and Buchner, et al., (1992)Anal. Biochem., 205: 263-270). Debinski, et al., for example, describethe denaturation and reduction of inclusion body proteins inguanidine-DTE. The proteins can be refolded in a redox buffercontaining, including but not limited to, oxidized glutathione andL-arginine. Refolding reagents can be flowed or otherwise moved intocontact with the one or more polypeptide or other expression product, orvice-versa.

In the case of prokaryotic production of FGF-21 polypeptide, the FGF-21polypeptide thus produced may be misfolded and thus lacks or has reducedbiological activity. The bioactivity of the protein may be restored by“refolding”. In general, misfolded FGF-21 polypeptide is refolded bysolubilizing (where the FGF-21 polypeptide is also insoluble), unfoldingand reducing the polypeptide chain using, for example, one or morechaotropic agents (e.g. urea and/or guanidine) and a reducing agentcapable of reducing disulfide bonds (e.g. dithiothreitol, DTT or2-mercaptoethanol, 2-ME). At a moderate concentration of chaotrope, anoxidizing agent is then added (e.g., oxygen, cystine or cystamine),which allows the reformation of disulfide bonds. FGF-21 polypeptide maybe refolded using standard methods known in the art, such as thosedescribed in U.S. Pat. Nos. 4,511,502, 4,511,503, and 4,512,922, whichare incorporated by reference herein. The FGF-21 polypeptide may also becofolded with other proteins to form heterodimers or heteromultimers.

After refolding, the FGF-21 may be further purified. Purification ofFGF-21 may be accomplished using a variety of techniques known to thoseof ordinary skill in the art, including hydrophobic interactionchromatography, size exclusion chromatography, ion exchangechromatography, reverse-phase high performance liquid chromatography,affinity chromatography, and the like or any combination thereof.Additional purification may also include a step of drying orprecipitation of the purified protein.

After purification, FGF-21 may be exchanged into different buffersand/or concentrated by any of a variety of methods known to the art,including, but not limited to, diafiltration and dialysis. FGF-21 thatis provided as a single purified protein may be subject to aggregationand precipitation.

The purified FGF-21 may be at least 90% pure (as measured by reversephase high performance liquid chromatography, RP-HPLC, or sodium dodecylsulfate-polyacrylamide gel electrophoresis, SDS-PAGE) or at least 95%pure, or at least 98% pure, or at least 99% or greater pure. Regardlessof the exact numerical value of the purity of the FGF-21, the FGF-21 issufficiently pure for use as a pharmaceutical product or for furtherprocessing, such as conjugation with a water soluble polymer such asPEG.

Certain FGF-21 molecules may be used as therapeutic agents in theabsence of other active ingredients or proteins (other than excipients,carriers, and stabilizers, serum albumin and the like), or they may becomplexed with another protein or a polymer.

General Purification Methods Any one of a variety of isolation steps maybe performed on the cell lysate, extract, culture medium, inclusionbodies, periplasmic space of the host cells, cytoplasm of the hostcells, or other material, comprising FGF-21 polypeptide or on any FGF-21polypeptide mixtures resulting from any isolation steps including, butnot limited to, affinity chromatography, ion exchange chromatography,hydrophobic interaction chromatography, gel filtration chromatography,high performance liquid chromatography (“HPLC”), reversed phase-HPLC(“RP-HPLC”), expanded bed adsorption, or any combination and/orrepetition thereof and in any appropriate order.

Equipment and other necessary materials used in performing thetechniques described herein are commercially available. Pumps, fractioncollectors, monitors, recorders, and entire systems are available from,for example, Applied Biosystems (Foster City, Calif.), Bio-RadLaboratories, Inc. (Hercules, Calif.), and Amersham Biosciences, Inc.(Piscataway, N.J.). Chromatographic materials including, but not limitedto, exchange matrix materials, media, and buffers are also availablefrom such companies.

Equilibration, and other steps in the column chromatography processesdescribed herein such as washing and elution, may be more rapidlyaccomplished using specialized equipment such as a pump. Commerciallyavailable pumps include, but are not limited to, HILOAD® Pump P-50,Peristaltic Pump P-1, Pump P-901, and Pump P-903 (Amersham Biosciences,Piscataway, N.J.).

Examples of fraction collectors include RediFrac Fraction Collector,FRAC-100 and FRAC-200 Fraction Collectors, and SUPERFRAC® FractionCollector (Amersham Biosciences, Piscataway, N.J.). Mixers are alsoavailable to form pH and linear concentration gradients. Commerciallyavailable mixers include Gradient Mixer GM-1 and In-Line Mixers(Amersham Biosciences, Piscataway, N.J.).

The chromatographic process may be monitored using any commerciallyavailable monitor. Such monitors may be used to gather information likeUV, pH, and conductivity. Examples of detectors include Monitor UV-1,UVICORD® S II, Monitor UV-M II, Monitor UV-900, Monitor UPC-900, MonitorpH/C-900, and Conductivity Monitor (Amersham Biosciences, Piscataway,N.J.). Indeed, entire systems are commercially available including thevarious AKTA® systems from Amersham Biosciences (Piscataway, N.J.).

In one embodiment of the present invention, for example, the FGF-21polypeptide may be reduced and denatured by first denaturing theresultant purified FGF-21 polypeptide in urea, followed by dilution intoTRIS buffer containing a reducing agent (such as DTT) at a suitable pH.In another embodiment, the FGF-21 polypeptide is denatured in urea in aconcentration range of between about 2 M to about 9 M, followed bydilution in TRIS buffer at a pH in the range of about 5.0 to about 8.0.The refolding mixture of this embodiment may then be incubated. In oneembodiment, the refolding mixture is incubated at room temperature forfour to twenty-four hours. The reduced and denatured FGF-21 polypeptidemixture may then be further isolated or purified.

As stated herein, the pH of the first FGF-21 polypeptide mixture may beadjusted prior to performing any subsequent isolation steps. Inaddition, the first FGF-21 polypeptide mixture or any subsequent mixturethereof may be concentrated using techniques known in the art. Moreover,the elution buffer comprising the first FGF-21 polypeptide mixture orany subsequent mixture thereof may be exchanged for a buffer suitablefor the next isolation step using techniques known to those of ordinaryskill in the art.

Ion Exchange Chromatography In one embodiment, and as an optional,additional step, ion exchange chromatography may be performed on thefirst FGF-21 polypeptide mixture. See generally ION EXCHANGECHROMATOGRAPHY: PRINCIPLES AND METHODS (Cat. No. 18-1114-21, AmershamBiosciences (Piscataway, N.J.)). Commercially available ion exchangecolumns include HITRAP®, HIPREP®, and HILOAD® Columns (AmershamBiosciences, Piscataway, N.J.). Such columns utilize strong anionexchangers such as Q SEPHAROSE® Fast Flow, Q SEPHAROSE® HighPerformance, and Q SEPHAROSE® XL; strong cation exchangers such as SPSEPHAROSE® High Performance, SP SEPHAROSE® Fast Flow, and SP SEPHAROSE®XL; weak anion exchangers such as DEAE SEPHAROSE® Fast Flow; and weakcation exchangers such as CM SEPHAROSE® Fast Flow (Amersham Biosciences,Piscataway, N.J.). Anion or cation exchange column chromatography may beperformed on the FGF-21 polypeptide at any stage of the purificationprocess to isolate substantially purified FGF-21 polypeptide. The cationexchange chromatography step may be performed using any suitable cationexchange matrix. Useful cation exchange matrices include, but are notlimited to, fibrous, porous, non-porous, microgranular, beaded, orcross-linked cation exchange matrix materials. Such cation exchangematrix materials include, but are not limited to, cellulose, agarose,dextran, polyacrylate, polyvinyl, polystyrene, silica, polyether, orcomposites of any of the foregoing.

The cation exchange matrix may be any suitable cation exchangerincluding strong and weak cation exchangers. Strong cation exchangersmay remain ionized over a wide pH range and thus, may be capable ofbinding FGF-21 over a wide pH range. Weak cation exchangers, however,may lose ionization as a function of pH. For example, a weak cationexchanger may lose charge when the pH drops below about pH 4 or pH 5.Suitable strong cation exchangers include, but are not limited to,charged functional groups such as sulfopropyl (SP), methyl sulfonate(S), or sulfoethyl (SE). The cation exchange matrix may be a strongcation exchanger, preferably having an FGF-21 binding pH range of about2.5 to about 6.0. Alternatively, the strong cation exchanger may have anFGF-21 binding pH range of about pH 2.5 to about pH 5.5. The cationexchange matrix may be a strong cation exchanger having an FGF-21binding pH of about 3.0. Alternatively, the cation exchange matrix maybe a strong cation exchanger, preferably having an FGF-21 binding pHrange of about 6.0 to about 8.0. The cation exchange matrix may be astrong cation exchanger preferably having an FGF-21 binding pH range ofabout 8.0 to about 12.5. Alternatively, the strong cation exchanger mayhave an FGF-21 binding pH range of about pH 8.0 to about pH 12.0.

Prior to loading the FGF-21, the cation exchange matrix may beequilibrated, for example, using several column volumes of a dilute,weak acid, e.g., four column volumes of 20 mM acetic acid, pH 3.Following equilibration, the FGF-21 may be added and the column may bewashed one to several times, prior to elution of substantially purifiedFGF-21, also using a weak acid solution such as a weak acetic acid orphosphoric acid solution. For example, approximately 2-4 column volumesof 20 mM acetic acid, pH 3, may be used to wash the column. Additionalwashes using, e.g., 2-4 column volumes of 0.05 M sodium acetate, pH 5.5,or 0.05 M sodium acetate mixed with 0.1 M sodium chloride, pH 5.5, mayalso be used. Alternatively, using methods known in the art, the cationexchange matrix may be equilibrated using several column volumes of adilute, weak base.

Alternatively, substantially purified FGF-21 may be eluted by contactingthe cation exchanger matrix with a buffer having a sufficiently low pHor ionic strength to displace the FGF-21 from the matrix. The pH of theelution buffer may range from about pH 2.5 to about pH 6.0. Morespecifically, the pH of the elution buffer may range from about pH 2.5to about pH 5.5, about pH 2.5 to about pH 5.0. The elution buffer mayhave a pH of about 3.0. In addition, the quantity of elution buffer mayvary widely and will generally be in the range of about 2 to about 10column volumes.

Following adsorption of the FGF-21 polypeptide to the cation exchangermatrix, substantially purified FGF-21 polypeptide may be eluted bycontacting the matrix with a buffer having a sufficiently high pH orionic strength to displace the FGF-21 polypeptide from the matrix.Suitable buffers for use in high pH elution of substantially purifiedFGF-21 polypeptide may include, but not limited to, citrate, phosphate,formate, acetate, HEPES, and IVIES buffers ranging in concentration fromat least about 5 mM to at least about 100 mM.

Reverse-Phase Chromatography RP-HPLC may be performed to purify proteinsfollowing suitable protocols that are known to those of ordinary skillin the art. See, e.g., Pearson et al., ANAL BIOCHEM. (1982) 124:217-230(1982); Rivier et al., J. CHROM. (1983) 268:112-119; Kunitani et al., J.CHROM. (1986) 359:391-402. RP-HPLC may be performed on the FGF-21polypeptide to isolate substantially purified FGF-21 polypeptide. Inthis regard, silica derivatized resins with alkyl functionalities with awide variety of lengths, including, but not limited to, at least aboutC₃ to at least about C₃₀, at least about C₃ to at least about C₂₀, or atleast about C₃ to at least about C₁₈, resins may be used. Alternatively,a polymeric resin may be used. For example, TosoHaas AmberchromeCG1000sd resin may be used, which is a styrene polymer resin. Cyano orpolymeric resins with a wide variety of alkyl chain lengths may also beused. Furthermore, the RP-HPLC column may be washed with a solvent suchas ethanol. The Source RP column is another example of a RP-HPLC column.

A suitable elution buffer containing an ion pairing agent and an organicmodifier such as methanol, isopropanol, tetrahydrofuran, acetonitrile orethanol, may be used to elute the FGF-21 polypeptide from the RP-HPLCcolumn. The most commonly used ion pairing agents include, but are notlimited to, acetic acid, formic acid, perchloric acid, phosphoric acid,trifluoroacetic acid, heptafluorobutyric acid, triethylamine,tetramethylammonium, tetrabutylammonium, and triethylammonium acetate.Elution may be performed using one or more gradients or isocraticconditions, with gradient conditions preferred to reduce the separationtime and to decrease peak width. Another method involves the use of twogradients with different solvent concentration ranges. Examples ofsuitable elution buffers for use herein may include, but are not limitedto, ammonium acetate and acetonitrile solutions.

Hydrophobic Interaction Chromatography Purification TechniquesHydrophobic interaction chromatography (HIC) may be performed on theFGF-21 polypeptide. See generally HYDROPHOBIC INTERACTION CHROMATOGRAPHYHANDBOOK: PRINCIPLES AND METHODS (Cat. No. 18-1020-90, AmershamBiosciences (Piscataway, N.J.) which is incorporated by referenceherein. Suitable HIC matrices may include, but are not limited to,alkyl- or aryl-substituted matrices, such as butyl-, hexyl-, octyl- orphenyl-substituted matrices including agarose, cross-linked agarose,sepharose, cellulose, silica, dextran, polystyrene, poly(methacrylate)matrices, and mixed mode resins, including but not limited to, apolyethyleneamine resin or a butyl- or phenyl-substitutedpoly(methacrylate) matrix. Commercially available sources forhydrophobic interaction column chromatography include, but are notlimited to, HITRAP®, HIPREP®, and HILOAD® columns (Amersham Biosciences,Piscataway, N.J.).

Briefly, prior to loading, the HIC column may be equilibrated usingstandard buffers known to those of ordinary skill in the art, such as anacetic acid/sodium chloride solution or HEPES containing ammoniumsulfate. Ammonium sulfate may be used as the buffer for loading the HICcolumn. After loading the FGF-21 polypeptide, the column may then washedusing standard buffers and conditions to remove unwanted materials butretaining the FGF-21 polypeptide on the HIC column. The FGF-21polypeptide may be eluted with about 3 to about 10 column volumes of astandard buffer, such as a HEPES buffer containing EDTA and lowerammonium sulfate concentration than the equilibrating buffer, or anacetic acid/sodium chloride buffer, among others. A decreasing linearsalt gradient using, for example, a gradient of potassium phosphate, mayalso be used to elute the FGF-21 molecules. The eluant may then beconcentrated, for example, by filtration such as diafiltration orultrafiltration. Diafiltration may be utilized to remove the salt usedto elute the FGF-21 polypeptide.

Other Purification Techniques Yet another isolation step using, forexample, gel filtration (GEL FILTRATION: PRINCIPLES AND METHODS (Cat.No. 18-1022-18, Amersham Biosciences, Piscataway, N.J.) which isincorporated by reference herein, hydroxyapatite chromatography(suitable matrices include, but are not limited to, HA-Ultrogel, HighResolution (Calbiochem), CHT Ceramic Hydroxyapatite (BioRad), Bio-GelHTP Hydroxyapatite (BioRad)), HPLC, expanded bed adsorption,ultrafiltration, diafiltration, lyophilization, and the like, may beperformed on the first FGF-21 polypeptide mixture or any subsequentmixture thereof, to remove any excess salts and to replace the bufferwith a suitable buffer for the next isolation step or even formulationof the final drug product.

The yield of FGF-21 polypeptide, including substantially purified FGF-21polypeptide, may be monitored at each step described herein usingtechniques known to those of ordinary skill in the art. Such techniquesmay also be used to assess the yield of substantially purified FGF-21polypeptide following the last isolation step. For example, the yield ofFGF-21 polypeptide may be monitored using any of several reverse phasehigh pressure liquid chromatography columns, having a variety of alkylchain lengths such as cyano RP-HPLC, C₁₈RP-HPLC; as well as cationexchange HPLC and gel filtration HPLC.

In specific embodiments of the present invention, the yield of FGF-21after each purification step may be at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, at least about99.9%, or at least about 99.99%, of the FGF-21 in the starting materialfor each purification step.

Purity may be determined using standard techniques, such as SDS-PAGE, orby measuring FGF-21 polypeptide using Western blot and ELISA assays. Forexample, polyclonal antibodies may be generated against proteinsisolated from negative control yeast fermentation and the cationexchange recovery. The antibodies may also be used to probe for thepresence of contaminating host cell proteins.

RP-HPLC material Vydac C4 (Vydac) consists of silica gel particles, thesurfaces of which carry C4-alkyl chains. The separation of FGF-21polypeptide from the proteinaceous impurities is based on differences inthe strength of hydrophobic interactions. Elution is performed with anacetonitrile gradient in diluted trifluoroacetic acid. Preparative HPLCis performed using a stainless steel column (filled with 2.8 to 3.2liter of Vydac C4 silicagel). The Hydroxyapatite Ultrogel eluate isacidified by adding trifluoroacetic acid and loaded onto the Vydac C4column. For washing and elution an acetonitrile gradient in dilutedtrifluoroacetic acid is used. Fractions are collected and immediatelyneutralized with phosphate buffer. The FGF-21 polypeptide fractionswhich are within the IPC limits are pooled.

DEAE Sepharose (Pharmacia) material consists of diethylaminoethyl(DEAE)-groups which are covalently bound to the surface of Sepharosebeads. The binding of FGF-21 polypeptide to the DEAE groups is mediatedby ionic interactions. Acetonitrile and trifluoroacetic acid passthrough the column without being retained. After these substances havebeen washed off, trace impurities are removed by washing the column withacetate buffer at a low pH. Then the column is washed with neutralphosphate buffer and FGF-21 polypeptide is eluted with a buffer withincreased ionic strength. The column is packed with DEAE Sepharose fastflow. The column volume is adjusted to assure a FGF-21 polypeptide loadin the range of 3-10 mg FGF-21 polypeptide/ml gel. The column is washedwith water and equilibration buffer (sodium/potassium phosphate). Thepooled fractions of the HPLC eluate are loaded and the column is washedwith equilibration buffer. Then the column is washed with washing buffer(sodium acetate buffer) followed by washing with equilibration buffer.Subsequently, FGF-21 polypeptide is eluted from the column with elutionbuffer (sodium chloride, sodium/potassium phosphate) and collected in asingle fraction in accordance with the master elution profile. Theeluate of the DEAE Sepharose column is adjusted to the specifiedconductivity. The resulting drug substance is sterile filtered intoTeflon bottles and stored at −70° C.

Additional methods that may be employed include, but are not limited to,steps to remove endotoxins. Endotoxins are lipopoly-saccharides (LPSs)which are located on the outer membrane of Gram-negative host cells,such as, for example, Escherichia coli. Methods for reducing endotoxinlevels are known to one of ordinary skill in the art and include, butare not limited to, purification techniques using silica supports, glasspowder or hydroxyapatite, reverse-phase, affinity, size-exclusion,anion-exchange chromatography, hydrophobic interaction chromatography, acombination of these methods, and the like. Modifications or additionalmethods may be required to remove contaminants such as co-migratingproteins from the polypeptide of interest. Methods for measuringendotoxin levels are known to one of ordinary skill in the art andinclude, but are not limited to, Limulus Amebocyte Lysate (LAL) assays.The Endosafe™-PTS assay is a colorimetric, single tube system thatutilizes cartridges preloaded with LAL reagent, chromogenic substrate,and control standard endotoxin along with a handheld spectrophotometer.Alternate methods include, but are not limited to, a Kinetic LAL methodthat is turbidmetric and uses a 96 well format.

A wide variety of methods and procedures can be used to assess the yieldand purity of a FGF-21 protein comprising one or more non-naturallyencoded amino acids, including but not limited to, the Bradford assay,SDS-PAGE, silver stained SDS-PAGE, coomassie stained SDS-PAGE, massspectrometry (including but not limited to, MALDI-TOF) and other methodsfor characterizing proteins known to one of ordinary skill in the art.

Additional methods include, but are not limited to: SDS-PAGE coupledwith protein staining methods, immunoblotting, matrix assisted laserdesorption/ionization-mass spectrometry (MALDI-MS), liquidchromatography/mass spectrometry, isoelectric focusing, analytical anionexchange, chromatofocusing, and circular dichroism.

VIII. Expression in Alternate Systems

Several strategies have been employed to introduce unnatural amino acidsinto proteins in non-recombinant host cells, mutagenized host cells, orin cell-free systems. These systems are also suitable for use in makingthe FGF-21 polypeptides of the present invention. Derivatization ofamino acids with reactive side-chains such as Lys, Cys and Tyr resultedin the conversion of lysine to N²-acetyl-lysine. Chemical synthesis alsoprovides a straightforward method to incorporate unnatural amino acids.With the recent development of enzymatic ligation and native chemicalligation of peptide fragments, it is possible to make larger proteins.See, e.g., P. E. Dawson and S. B. H. Kent, Annu. Rev. Biochem, 69:923(2000). Chemical peptide ligation and native chemical ligation aredescribed in U.S. Pat. No. 6,184,344, U.S. Patent Publication No.2004/0138412, U.S. Patent Publication No. 2003/0208046, WO 02/098902,and WO 03/042235, which are incorporated by reference herein. A generalin vitro biosynthetic method in which a suppressor tRNA chemicallyacylated with the desired unnatural amino acid is added to an in vitroextract capable of supporting protein biosynthesis, has been used tosite-specifically incorporate over 100 unnatural amino acids into avariety of proteins of virtually any size. See, e.g., V. W. Cornish, D.Mendel and P. G. Schultz, Angew. Chem. Int. Ed. Engl., 1995, 34:621(1995); C. J. Noren, S.J. Anthony-Cahill, M.C. Griffith, P.G. Schultz, Ageneral method for site-specific incorporation of unnatural amino acidsinto proteins, Science 244:182-188 (1989); and, J.D. Bain, C.G. Glabe,T.A. Dix, A.R. Chamberlin, E.S. Diala, Biosynthetic site-specificincorporation of a non-natural amino acid into a polypeptide, J. Am.Chem. Soc. 111:8013-8014 (1989). A broad range of functional groups hasbeen introduced into proteins for studies of protein stability, proteinfolding, enzyme mechanism, and signal transduction.

An in vivo method, termed selective pressure incorporation, wasdeveloped to exploit the promiscuity of wild-type synthetases. See,e.g., N. Budisa, C. Minks, S. Alefelder, W. Wenger, F. M. Dong, L.Moroder and R. Huber, FASEB J., 13:41 (1999). An auxotrophic strain, inwhich the relevant metabolic pathway supplying the cell with aparticular natural amino acid is switched off, is grown in minimal mediacontaining limited concentrations of the natural amino acid, whiletranscription of the target gene is repressed. At the onset of astationary growth phase, the natural amino acid is depleted and replacedwith the unnatural amino acid analog. Induction of expression of therecombinant protein results in the accumulation of a protein containingthe unnatural analog. For example, using this strategy, o, m andp-fluorophenylalanines have been incorporated into proteins, and exhibittwo characteristic shoulders in the UV spectrum which can be easilyidentified, see, e.g., C. Minks, R. Huber, L. Moroder and N. Budisa,Anal. Biochem., 284:29 (2000); trifluoromethionine has been used toreplace methionine in bacteriophage T₄ lysozyme to study its interactionwith chitooligosaccharide ligands by ¹⁹F NMR, see, e.g., H. Duewel, E.Daub, V. Robinson and J. F. Honek, Biochemistry, 36:3404 (1997); andtrifluoroleucine has been incorporated in place of leucine, resulting inincreased thermal and chemical stability of a leucine-zipper protein.See, e.g., Y. Tang, G. Ghirlanda, W. A. Petka, T. Nakajima, W. F.DeGrado and D. A. Tirrell, Angew. Chem. Int. Ed. Engl., 40:1494 (2001).Moreover, selenomethionine and telluromethionine are incorporated intovarious recombinant proteins to facilitate the solution of phases inX-ray crystallography. See, e.g., W. A. Hendrickson, J. R. Horton and D.M. Lemaster, EMBO J., 9:1665 (1990); J. O. Boles, K. Lewinski, M.Kunkle, J. D. Odom, B. Dunlap, L. Lebioda and M. Hatada, Nat. Struct.Biol., 1:283 (1994); N. Budisa, B. Steipe, P. Demange, C. Eckerskorn, J.Kellermann and R. Huber, Eur. J. Biochem., 230:788 (1995); and, N.Budisa, W. Karnbrock, S. Steinbacher, A. Humm, L. Prade, T. Neuefeind,L. Moroder and R. Huber, J. Mol. Biol., 270:616 (1997). Methionineanalogs with alkene or alkyne functionalities have also beenincorporated efficiently, allowing for additional modification ofproteins by chemical means. See, e.g., J. C. van Hest and D. A. Tirrell,FEBS Lett., 428:68 (1998); J. C. van Hest, K. L. Kiick and D. A.Tirrell, J. Am. Chem. Soc., 122:1282 (2000); and, K. L. Kiick and D. A.Tirrell, Tetrahedron, 56:9487 (2000); U.S. Pat. No. 6,586,207; U.S.Patent Publication 2002/0042097, which are incorporated by referenceherein.

The success of this method depends on the recognition of the unnaturalamino acid analogs by aminoacyl-tRNA synthetases, which, in general,require high selectivity to insure the fidelity of protein translation.One way to expand the scope of this method is to relax the substratespecificity of aminoacyl-tRNA synthetases, which has been achieved in alimited number of cases. For example, replacement of Ala²⁹⁴ by Gly inEscherichia coli phenylalanyl-tRNA synthetase (PheRS) increases the sizeof substrate binding pocket, and results in the acylation of tRNAPhe byp-Cl-phenylalanine (p-Cl-Phe). See, M. Ibba, P. Kast and H. Hennecke,Biochemistry, 33:7107 (1994). An Escherichia coli strain harboring thismutant PheRS allows the incorporation of p-Cl-phenylalanine orp-Br-phenylalanine in place of phenylalanine. See, e.g., M. Ibba and H.Hennecke, FEBS Lett., 364:272 (1995); and, N. Sharma, R. Furter, P. Kastand D. A. Tirrell, FEBS Lett., 467:37 (2000). Similarly, a pointmutation Phe130Ser near the amino acid binding site of Escherichia colityrosyl-tRNA synthetase was shown to allow azatyrosine to beincorporated more efficiently than tyrosine. See, F. Hamano-Takaku, T.Iwama, S. Saito-Yano, K. Takaku, Y. Monden, M. Kitabatake, D. Soll andS. Nishimura, J. Biol. Chem., 275:40324 (2000).

Another strategy to incorporate unnatural amino acids into proteins invivo is to modify synthetases that have proofreading mechanisms. Thesesynthetases cannot discriminate and therefore activate amino acids thatare structurally similar to the cognate natural amino acids. This erroris corrected at a separate site, which deacylates the mischarged aminoacid from the tRNA to maintain the fidelity of protein translation. Ifthe proofreading activity of the synthetase is disabled, structuralanalogs that are misactivated may escape the editing function and beincorporated. This approach has been demonstrated recently with thevalyl-tRNA synthetase (ValRS). See, V. Doring, H. D. Mootz, L. A.Nangle, T. L. Hendrickson, V. de Crecy-Lagard, P. Schimmel and P.Marliere, Science, 292:501 (2001). ValRS can misaminoacylate tRNAValwith Cys, Thr, or aminobutyrate (Abu); these noncognate amino acids aresubsequently hydrolyzed by the editing domain. After random mutagenesisof the Escherichia coli chromosome, a mutant Escherichia coli strain wasselected that has a mutation in the editing site of ValRS. Thisedit-defective ValRS incorrectly charges tRNAVal with Cys. Because Abusterically resembles Cys (—SH group of Cys is replaced with —CH₃ inAbu), the mutant ValRS also incorporates Abu into proteins when thismutant Escherichia coli strain is grown in the presence of Abu. Massspectrometric analysis shows that about 24% of valines are replaced byAbu at each valine position in the native protein.

Solid-phase synthesis and semisynthetic methods have also allowed forthe synthesis of a number of proteins containing novel amino acids. Forexample, see the following publications and references cited within,which are as follows: Crick, F.H.C., Barrett, L. Brenner, S.Watts-Tobin, R. General nature of the genetic code for proteins. Nature,192:1227-1232 (1961); Hofmann, K., Bohn, H. Studies on polypeptides.XXVI. The effect of pyrazole-imidazole replacements on the S-proteinactivating potency of an S-peptide fragment, J. Am Chem,88(24):5914-5919 (1966); Kaiser, E.T. Synthetic approaches tobiologically active peptides and proteins including enyzmes, Acc ChemRes, 22:47-54 (1989); Nakatsuka, T., Sasaki, T., Kaiser, E.T. Peptidesegment coupling catalyzed by the semisynthetic enzyme thiosubtilisin, JAm Chem Soc, 109:3808-3810 (1987); Schnolzer, M., Kent, S B H.Constructing proteins by dovetailing unprotected synthetic peptides:backbone-engineered HIV protease, Science, 256(5054):221-225 (1992);Chaiken, I.M. Semisynthetic peptides and proteins, CRC Crit Rev Biochem,11(3):255-301 (1981); Offord, R.E. Protein engineering by chemicalmeans? Protein Eng., 1(3):151-157 (1987); and, Jackson, D.Y., Burnier,J., Quan, C., Stanley, M., Tom, J., Wells, J.A. A Designed PeptideLigase for Total Synthesis of Ribonuclease A with Unnatural CatalyticResidues, Science, 266(5183):243 (1994).

Chemical modification has been used to introduce a variety of unnaturalside chains, including cofactors, spin labels and oligonucleotides intoproteins in vitro. See, e.g., Corey, D.R., Schultz, P.G. Generation of ahybrid sequence-specific single-stranded deoxyribonuclease, Science,238(4832):1401-1403 (1987); Kaiser, E.T., Lawrence D.S., Rokita, S.E.The chemical modification of enzymatic specificity, Annu Rev Biochem,54:565-595 (1985); Kaiser, E.T., Lawrence, D.S. Chemical mutation ofenyzme active sites, Science, 226(4674):505-511 (1984); Neet, K.E.,Nanci A, Koshland, D.E. Properties of thiol-subtilisin, J Biol. Chem,243(24):6392-6401 (1968); Polgar, L. et M.L. Bender. A new enzymecontaining a synthetically formed active site. Thiol-subtilisin. J. AmChem Soc, 88:3153-3154 (1966); and, Pollack, S.J., Nakayama, G. Schultz,P.G. Introduction of nucleophiles and spectroscopic probes into antibodycombining sites, Science, 242(4881):1038-1040 (1988).

Alternatively, biosynthetic methods that employ chemically modifiedaminoacyl-tRNAs have been used to incorporate several biophysical probesinto proteins synthesized in vitro. See the following publications andreferences cited within: Brunner, J. New Photolabeling and crosslinkingmethods, Annu. Rev Biochem, 62:483-514 (1993); and, Krieg, U.C., Walter,P., Hohnson, A.E. Photocrosslinking of the signal sequence of nascentpreprolactin of the 54-kilodalton polypeptide of the signal recognitionparticle, Proc. Natl. Acad. Sci, 83(22):8604-8608 (1986).

Previously, it has been shown that unnatural amino acids can besite-specifically incorporated into proteins in vitro by the addition ofchemically aminoacylated suppressor tRNAs to protein synthesis reactionsprogrammed with a gene containing a desired amber nonsense mutation.Using these approaches, one can substitute a number of the common twentyamino acids with close structural homologues, e.g., fluorophenylalaninefor phenylalanine, using strains auxotropic for a particular amino acid.See, e.g., Noren, C.J., Anthony-Cahill, Griffith, M.C., Schultz, P.G. Ageneral method for site-specific incorporation of unnatural amino acidsinto proteins, Science, 244: 182-188 (1989); M. W. Nowak, et al.,Science 268:439-42 (1995); Bain, J.D., Glabe, C.G., Dix, T.A.,Chamberlin, A.R., Diala, E.S. Biosynthetic site-specific Incorporationof a non-natural amino acid into a polypeptide, J. Am Chem Soc,111:8013-8014 (1989); N. Budisa et al., FASEB J. 13:41-51 (1999);Ellman, J.A., Mendel, D., Anthony-Cahill, S., Noren, C.J., Schultz, P.G.Biosynthetic method for introducing unnatural amino acidssite-specifically into proteins, Methods in Enz., vol. 202, 301-336(1992); and, Mendel, D., Cornish, V.W. & Schultz, P.G. Site-DirectedMutagenesis with an Expanded Genetic Code, Annu Rev Biophys. BiomolStruct. 24, 435-62 (1995).

For example, a suppressor tRNA was prepared that recognized the stopcodon UAG and was chemically aminoacylated with an unnatural amino acid.Conventional site-directed mutagenesis was used to introduce the stopcodon TAG, at the site of interest in the protein gene. See, e.g.,Sayers, J.R., Schmidt, W. Eckstein, F. 5′-3′ Exonucleases inphosphorothioate-based olignoucleotide-directed mutagensis, NucleicAcids Res, 16(3):791-802 (1988). When the acylated suppressor tRNA andthe mutant gene were combined in an in vitro transcription/translationsystem, the unnatural amino acid was incorporated in response to the UAGcodon which gave a protein containing that amino acid at the specifiedposition. Experiments using [³H]-Phe and experiments with a-hydroxyacids demonstrated that only the desired amino acid is incorporated atthe position specified by the UAG codon and that this amino acid is notincorporated at any other site in the protein. See, e.g., Noren, et al,supra; Kobayashi et al., (2003) Nature Structural Biology 10(6):425-432;and, Ellman, J.A., Mendel, D., Schultz, P.G. Site-specific incorporationof novel backbone structures into proteins, Science, 255(5041):197-200(1992).

A tRNA may be aminoacylated with a desired amino acid by any method ortechnique, including but not limited to, chemical or enzymaticaminoacylation.

Aminoacylation may be accomplished by aminoacyl tRNA synthetases or byother enzymatic molecules, including but not limited to, ribozymes. Theterm “ribozyme” is interchangeable with “catalytic RNA.” Cech andcoworkers (Cech, 1987, Science, 236:1532-1539; McCorkle et al., 1987,Concepts Biochem. 64:221-226) demonstrated the presence of naturallyoccurring RNAs that can act as catalysts (ribozymes). However, althoughthese natural RNA catalysts have only been shown to act on ribonucleicacid substrates for cleavage and splicing, the recent development ofartificial evolution of ribozymes has expanded the repertoire ofcatalysis to various chemical reactions. Studies have identified RNAmolecules that can catalyze aminoacyl-RNA bonds on their own(2′)₃′-termini (Illangakekare et al., 1995 Science 267:643-647), and anRNA molecule which can transfer an amino acid from one RNA molecule toanother (Lohse et al., 1996, Nature 381:442-444).

U.S. Patent Application Publication 2003/0228593, which is incorporatedby reference herein, describes methods to construct ribozymes and theiruse in aminoacylation of tRNAs with naturally encoded and non-naturallyencoded amino acids. Substrate-immobilized forms of enzymatic moleculesthat can aminoacylate tRNAs, including but not limited to, ribozymes,may enable efficient affinity purification of the aminoacylatedproducts. Examples of suitable substrates include agarose, sepharose,and magnetic beads. The production and use of a substrate-immobilizedform of ribozyme for aminoacylation is described in Chemistry andBiology 2003, 10:1077-1084 and U.S. Patent Application Publication2003/0228593, which are incorporated by reference herein.

Chemical aminoacylation methods include, but are not limited to, thoseintroduced by Hecht and coworkers (Hecht, S. M. Acc. Chem. Res. 1992,25, 545; Heckler, T. G.; Roesser, J. R.; Xu, C.; Chang, P.; Hecht, S. M.Biochemistry 1988, 27, 7254; Hecht, S. M.; Alford, B. L.; Kuroda, Y.;Kitano, S. J. Biol. Chem. 1978, 253, 4517) and by Schultz, Chamberlin,Dougherty and others (Cornish, V. W.; Mendel, D.; Schultz, P. G. Angew.Chem. Int. Ed. Engl. 1995, 34, 621; Robertson, S. A.; Ellman, J. A.;Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 2722; Noren, C. J.;Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G. Science 1989,244, 182; Bain, J. D.; Glabe, C. G.; Dix, T. A.; Chamberlin, A. R. J.Am. Chem. Soc. 1989, 111, 8013; Bain, J. D. et al. Nature 1992, 356,537; Gallivan, J. P.; Lester, H. A.; Dougherty, D. A. Chem. Biol. 1997,4, 740; Turcatti, et al. J. Biol. Chem. 1996, 271, 19991; Nowak, M. W.et al. Science, 1995, 268, 439; Saks, M. E. et al. J. Biol. Chem. 1996,271, 23169; Hohsaka, T. et al. J. Am. Chem. Soc. 1999, 121, 34), whichare incorporated by reference herein, to avoid the use of synthetases inaminoacylation. Such methods or other chemical aminoacylation methodsmay be used to aminoacylate tRNA molecules.

Methods for generating catalytic RNA may involve generating separatepools of randomized ribozyme sequences, performing directed evolution onthe pools, screening the pools for desirable aminoacylation activity,and selecting sequences of those ribozymes exhibiting desiredaminoacylation activity.

Ribozymes can comprise motifs and/or regions that facilitate acylationactivity, such as a GGU motif and a U-rich region. For example, it hasbeen reported that U-rich regions can facilitate recognition of an aminoacid substrate, and a GGU-motif can form base pairs with the 3′ terminiof a tRNA. In combination, the GGU and motif and U-rich regionfacilitate simultaneous recognition of both the amino acid and tRNAsimultaneously, and thereby facilitate aminoacylation of the 3′ terminusof the tRNA.

Ribozymes can be generated by in vitro selection using a partiallyrandomized r24mini conjugated with tRNA^(A)sncccG, followed bysystematic engineering of a consensus sequence found in the activeclones. An exemplary ribozyme obtained by this method is termed “Fx3ribozyme” and is described in U.S. Pub. App. No. 2003/0228593, thecontents of which is incorporated by reference herein, acts as aversatile catalyst for the synthesis of various aminoacyl-tRNAs chargedwith cognate non-natural amino acids.

Immobilization on a substrate may be used to enable efficient affinitypurification of the aminoacylated tRNAs. Examples of suitable substratesinclude, but are not limited to, agarose, sepharose, and magnetic beads.Ribozymes can be immobilized on resins by taking advantage of thechemical structure of RNA, such as the 3′-cis-diol on the ribose of RNAcan be oxidized with periodate to yield the corresponding dialdehyde tofacilitate immobilization of the RNA on the resin. Various types ofresins can be used including inexpensive hydrazide resins whereinreductive amination makes the interaction between the resin and theribozyme an irreversible linkage. Synthesis of aminoacyl-tRNAs can besignificantly facilitated by this on-column aminoacylation technique.Kourouklis et al. Methods 2005; 36:239-4 describe a column-basedaminoacylation system.

Isolation of the aminoacylated tRNAs can be accomplished in a variety ofways. One suitable method is to elute the aminoacylated tRNAs from acolumn with a buffer such as a sodium acetate solution with 10 mM EDTA,a buffer containing 50 mMN-(2-hydroxyethyl)piperazine-N′-(3-propanesulfonic acid), 12.5 mM KCl,pH 7.0, 10 mM EDTA, or simply an EDTA buffered water (pH 7.0).

The aminoacylated tRNAs can be added to translation reactions in orderto incorporate the amino acid with which the tRNA was aminoacylated in aposition of choice in a polypeptide made by the translation reaction.Examples of translation systems in which the aminoacylated tRNAs of thepresent invention may be used include, but are not limited to celllysates. Cell lysates provide reaction components necessary for in vitrotranslation of a polypeptide from an input mRNA. Examples of suchreaction components include but are not limited to ribosomal proteins,rRNA, amino acids, tRNAs, GTP, ATP, translation initiation andelongation factors and additional factors associated with translation.Additionally, translation systems may be batch translations orcompartmentalized translation. Batch translation systems combinereaction components in a single compartment while compartmentalizedtranslation systems separate the translation reaction components fromreaction products that can inhibit the translation efficiency. Suchtranslation systems are available commercially.

Further, a coupled transcription/translation system may be used. Coupledtranscription/translation systems allow for both transcription of aninput DNA into a corresponding mRNA, which is in turn translated by thereaction components. An example of a commercially available coupledtranscription/translation is the Rapid Translation System (RTS, RocheInc.). The system includes a mixture containing E. coli lysate forproviding translational components such as ribosomes and translationfactors. Additionally, an RNA polymerase is included for thetranscription of the input DNA into an mRNA template for use intranslation. RTS can use compartmentalization of the reaction componentsby way of a membrane interposed between reaction compartments, includinga supply/waste compartment and a transcription/translation compartment.

Aminoacylation of tRNA may be performed by other agents, including butnot limited to, transferases, polymerases, catalytic antibodies,multi-functional proteins, and the like.

Stephan in Scientist 2005 Oct. 10; pages 30-33 describes additionalmethods to incorporate non-naturally encoded amino acids into proteins.Lu et al. in Mol Cell. 2001 Oct.; 8(4):759-69 describe a method in whicha protein is chemically ligated to a synthetic peptide containingunnatural amino acids (expressed protein ligation).

Microinjection techniques have also been use incorporate unnatural aminoacids into proteins. See, e.g., M. W. Nowak, P. C. Kearney, J. R.Sampson, M. E. Saks, C. G. Labarca, S. K. Silverman, W. G. Zhong, J.Thorson, J. N. Abelson, N. Davidson, P. G. Schultz, D. A. Dougherty andH. A. Lester, Science, 268:439 (1995); and, D. A. Dougherty, Curr. Opin.Chem. Biol., 4:645 (2000). A Xenopus oocyte was coinjected with two RNAspecies made in vitro: an mRNA encoding the target protein with a UAGstop codon at the amino acid position of interest and an ambersuppressor tRNA aminoacylated with the desired unnatural amino acid. Thetranslational machinery of the oocyte then inserts the unnatural aminoacid at the position specified by UAG. This method has allowed in vivostructure-function studies of integral membrane proteins, which aregenerally not amenable to in vitro expression systems. Examples includethe incorporation of a fluorescent amino acid into tachykininneurokinin-2 receptor to measure distances by fluorescence resonanceenergy transfer, see, e.g., G. Turcatti, K. Nemeth, M. D. Edgerton, U.Meseth, F. Talabot, M. Peitsch, J. Knowles, H. Vogel and A. Chollet, J.Biol. Chem., 271:19991 (1996); the incorporation of biotinylated aminoacids to identify surface-exposed residues in ion channels, see, e.g.,J. P. Gallivan, H. A. Lester and D. A. Dougherty, Chem. Biol., 4:739(1997); the use of caged tyrosine analogs to monitor conformationalchanges in an ion channel in real time, see, e.g., J. C. Miller, S. K.Silverman, P. M. England, D. A. Dougherty and H. A. Lester, Neuron,20:619 (1998); and, the use of alpha hydroxy amino acids to change ionchannel backbones for probing their gating mechanisms. See, e.g., P. M.England, Y. Zhang, D. A. Dougherty and H. A. Lester, Cell, 96:89 (1999);and, T. Lu, A. Y. Ting, J. Mainland, L. Y. Jan, P. G. Schultz and J.Yang, Nat. Neurosci., 4:239 (2001).

The ability to incorporate unnatural amino acids directly into proteinsin vivo offers a wide variety of advantages including but not limitedto, high yields of mutant proteins, technical ease, the potential tostudy the mutant proteins in cells or possibly in living organisms andthe use of these mutant proteins in therapeutic treatments anddiagnostic uses. The ability to include unnatural amino acids withvarious sizes, acidities, nucleophilicities, hydrophobicities, and otherproperties into proteins can greatly expand our ability to rationallyand systematically manipulate the structures of proteins, both to probeprotein function and create new proteins or organisms with novelproperties.

In one attempt to site-specifically incorporate para-F-Phe, a yeastamber suppressor tRNAPheCUA/phenylalanyl-tRNA synthetase pair was usedin a p-F-Phe resistant, Phe auxotrophic Escherichia coli strain. See,e.g., R. Furter, Protein Sci., 7:419 (1998).

It may also be possible to obtain expression of a FGF-21 polynucleotideof the present invention using a cell-free (in-vitro) translationalsystem. Translation systems may be cellular or cell-free, and may beprokaryotic or eukaryotic. Cellular translation systems include, but arenot limited to, whole cell preparations such as permeabilized cells orcell cultures wherein a desired nucleic acid sequence can be transcribedto mRNA and the mRNA translated. Cell-free translation systems arecommercially available and many different types and systems arewell-known. Examples of cell-free systems include, but are not limitedto, prokaryotic lysates such as Escherichia coli lysates, and eukaryoticlysates such as wheat germ extracts, insect cell lysates, rabbitreticulocyte lysates, rabbit oocyte lysates and human cell lysates.Eukaryotic extracts or lysates may be preferred when the resultingprotein is glycosylated, phosphorylated or otherwise modified becausemany such modifications are only possible in eukaryotic systems. Some ofthese extracts and lysates are available commercially (Promega; Madison,Wis.; Stratagene; La Jolla, Calif.; Amersham; Arlington Heights, Ill.;GIBCO/BRL; Grand Island, N.Y.). Membranous extracts, such as the caninepancreatic extracts containing microsomal membranes, are also availablewhich are useful for translating secretory proteins. In these systems,which can include either mRNA as a template (in-vitro translation) orDNA as a template (combined in-vitro transcription and translation), thein vitro synthesis is directed by the ribosomes. Considerable effort hasbeen applied to the development of cell-free protein expression systems.See, e.g., Kim, D.M. and J.R. Swartz, Biotechnology and Bioengineering,74:309-316 (2001); Kim, D.M. and J.R. Swartz, Biotechnology Letters, 22,1537-1542, (2000); Kim, D.M., and J.R. Swartz, Biotechnology Progress,16, 385-390, (2000); Kim, D.M., and J.R. Swartz, Biotechnology andBioengineering, 66, 180-188, (1999); and Patnaik, R. and J.R. Swartz,Biotechniques 24, 862-868, (1998); U.S. Pat. No. 6,337,191; U.S. PatentPublication No. 2002/0081660; WO 00/55353; WO 90/05785, which areincorporated by reference herein. Another approach that may be appliedto the expression of FGF-21 polypeptides comprising a non-naturallyencoded amino acid includes the mRNA-peptide fusion technique. See,e.g., R. Roberts and J. Szostak, Proc. Natl Acad. Sci. (USA)94:12297-12302 (1997); A. Frankel, et al., Chemistry & Biology10:1043-1050 (2003). In this approach, an mRNA template linked topuromycin is translated into peptide on the ribosome. If one or moretRNA molecules has been modified, non-natural amino acids can beincorporated into the peptide as well. After the last mRNA codon hasbeen read, puromycin captures the C-terminus of the peptide. If theresulting mRNA-peptide conjugate is found to have interesting propertiesin an in vitro assay, its identity can be easily revealed from the mRNAsequence. In this way, one may screen libraries of FGF-21 polypeptidescomprising one or more non-naturally encoded amino acids to identifypolypeptides having desired properties. More recently, in vitro ribosometranslations with purified components have been reported that permit thesynthesis of peptides substituted with non-naturally encoded aminoacids. See, e.g., A. Forster et al., Proc. Natl Acad. Sci. (USA)100:6353 (2003).

Reconstituted translation systems may also be used. Mixtures of purifiedtranslation factors have also been used successfully to translate mRNAinto protein as well as combinations of lysates or lysates supplementedwith purified translation factors such as initiation factor-1 (IF-1),IF-2, IF-3 (a or (3), elongation factor T (EF-Tu), or terminationfactors. Cell-free systems may also be coupled transcription/translationsystems wherein DNA is introduced to the system, transcribed into mRNAand the mRNA translated as described in Current Protocols in MolecularBiology (F. M. Ausubel et al. editors, Wiley Interscience, 1993), whichis hereby specifically incorporated by reference. RNA transcribed ineukaryotic transcription system may be in the form of heteronuclear RNA(hnRNA) or 5′-end caps (7-methyl guanosine) and 3′-end poly A tailedmature mRNA, which can be an advantage in certain translation systems.For example, capped mRNAs are translated with high efficiency in thereticulocyte lysate system.

IX. Macromolecular Polymers Coupled to FGF-21 Polypeptides

Various modifications to the non-natural amino acid polypeptidesdescribed herein can be effected using the compositions, methods,techniques and strategies described herein. These modifications includethe incorporation of further functionality onto the non-natural aminoacid component of the polypeptide, including but not limited to, alabel; a dye; a polymer; a water-soluble polymer; a derivative ofpolyethylene glycol; a photocrosslinker; a radionuclide; a cytotoxiccompound; a drug; an affinity label; a photoaffinity label; a reactivecompound; a resin; a second protein or polypeptide or polypeptideanalog; an antibody or antibody fragment; a metal chelator; a cofactor;a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; anantisense polynucleotide; a saccharide; a water-soluble dendrimer; acyclodextrin; an inhibitory ribonucleic acid; a biomaterial; ananoparticle; a spin label; a fluorophore, a metal-containing moiety; aradioactive moiety; a novel functional group; a group that covalently ornoncovalently interacts with other molecules; a photocaged moiety; anactinic radiation excitable moiety; a photoisomerizable moiety; biotin;a derivative of biotin; a biotin analogue; a moiety incorporating aheavy atom; a chemically cleavable group; a photocleavable group; anelongated side chain; a carbon-linked sugar; a redox-active agent; anamino thioacid; a toxic moiety; an isotopically labeled moiety; abiophysical probe; a phosphorescent group; a chemiluminescent group; anelectron dense group; a magnetic group; an intercalating group; achromophore; an energy transfer agent; a biologically active agent; adetectable label; a small molecule; a quantum dot; a nanotransmitter; aradionucleotide; a radiotransmitter; a neutron-capture agent; or anycombination of the above, or any other desirable compound or substance.As an illustrative, non-limiting example of the compositions, methods,techniques and strategies described herein, the following descriptionwill focus on adding macromolecular polymers to the non-natural aminoacid polypeptide with the understanding that the compositions, methods,techniques and strategies described thereto are also applicable (withappropriate modifications, if necessary and for which one of skill inthe art could make with the disclosures herein) to adding otherfunctionalities, including but not limited to those listed above.

A wide variety of macromolecular polymers and other molecules can belinked to FGF-21 polypeptides of the present invention to modulatebiological properties of the FGF-21 polypeptide, and/or provide newbiological properties to the FGF-21 molecule. These macromolecularpolymers can be linked to the FGF-21 polypeptide via a naturally encodedamino acid, via a non-naturally encoded amino acid, or any functionalsubstituent of a natural or non-natural amino acid, or any substituentor functional group added to a natural or non-natural amino acid. Themolecular weight of the polymer may be of a wide range, including butnot limited to, between about 100 Da and about 100,000 Da or more. Themolecular weight of the polymer may be between about 100 Da and about100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da,55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da,700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In someembodiments, the molecular weight of the polymer is between about 100 Daand about 50,000 Da. In some embodiments, the molecular weight of thepolymer is between about 100 Da and about 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 1,000Da and about 40,000 Da. In some embodiments, the molecular weight of thepolymer is between about 5,000 Da and about 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 10,000Da and about 40,000 Da.

The present invention provides substantially homogenous preparations ofpolymer:protein conjugates. “Substantially homogenous” as used hereinmeans that polymer:protein conjugate molecules are observed to begreater than half of the total protein. The polymer:protein conjugatehas biological activity and the present “substantially homogenous”PEGylated FGF-21 polypeptide preparations provided herein are thosewhich are homogenous enough to display the advantages of a homogenouspreparation, e.g., ease in clinical application in predictability of lotto lot pharmacokinetics.

One may also choose to prepare a mixture of polymer:protein conjugatemolecules, and the advantage provided herein is that one may select theproportion of mono-polymer:protein conjugate to include in the mixture.Thus, if desired, one may prepare a mixture of various proteins withvarious numbers of polymer moieties attached (i.e., di-, tri-, tetra-,etc.) and combine said conjugates with the mono-polymer:proteinconjugate prepared using the methods of the present invention, and havea mixture with a predetermined proportion of mono-polymer:proteinconjugates.

The polymer selected may be water soluble so that the protein to whichit is attached does not precipitate in an aqueous environment, such as aphysiological environment. The polymer may be branched or unbranched.For therapeutic use of the end-product preparation, the polymer will bepharmaceutically acceptable.

Examples of polymers include but are not limited to polyalkyl ethers andalkoxy-capped analogs thereof (e.g., polyoxyethylene glycol,polyoxyethylene/propylene glycol, and methoxy or ethoxy-capped analogsthereof, especially polyoxyethylene glycol, the latter is also known aspolyethyleneglycol or PEG); polyvinylpyrrolidones; polyvinylalkylethers; polyoxazolines, polyalkyl oxazolines and polyhydroxyalkyloxazolines; polyacrylamides, polyalkyl acrylamides, and polyhydroxyalkylacrylamides (e.g., polyhydroxypropylmethacrylamide and derivativesthereof); polyhydroxyalkyl acrylates; polysialic acids and analogsthereof; hydrophilic peptide sequences; polysaccharides and theirderivatives, including dextran and dextran derivatives, e.g.,carboxymethyldextran, dextran sulfates, aminodextran; cellulose and itsderivatives, e.g., carboxymethyl cellulose, hydroxyalkyl celluloses;chitin and its derivatives, e.g., chitosan, succinyl chitosan,carboxymethylchitin, carboxymethylchitosan; hyaluronic acid and itsderivatives; starches; alginates; chondroitin sulfate; albumin; pullulanand carboxymethyl pullulan; polyaminoacids and derivatives thereof,e.g., polyglutamic acids, polylysines, polyaspartic acids,polyaspartamides; maleic anhydride copolymers such as: styrene maleicanhydride copolymer, divinylethyl ether maleic anhydride copolymer;polyvinyl alcohols; copolymers thereof; terpolymers thereof; mixturesthereof; and derivatives of the foregoing.

The proportion of polyethylene glycol molecules to protein moleculeswill vary, as will their concentrations in the reaction mixture. Ingeneral, the optimum ratio (in terms of efficiency of reaction in thatthere is minimal excess unreacted protein or polymer) may be determinedby the molecular weight of the polyethylene glycol selected and on thenumber of available reactive groups available. As relates to molecularweight, typically the higher the molecular weight of the polymer, thefewer number of polymer molecules which may be attached to the protein.Similarly, branching of the polymer should be taken into account whenoptimizing these parameters. Generally, the higher the molecular weight(or the more branches) the higher the polymer:protein ratio.

As used herein, and when contemplating PEG:FGF-21 polypeptideconjugates, the term “therapeutically effective amount” refers to anamount which gives the desired benefit to a patient. The amount willvary from one individual to another and will depend upon a number offactors, including the overall physical condition of the patient and theunderlying cause of the condition to be treated. The amount of FGF-21polypeptide used for therapy gives an acceptable rate of change andmaintains desired response at a beneficial level. A therapeuticallyeffective amount of the present compositions may be readily ascertainedby one of ordinary skill in the art using publicly available materialsand procedures.

The water soluble polymer may be any structural form including but notlimited to linear, forked or branched. Typically, the water solublepolymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG),but other water soluble polymers can also be employed. By way ofexample, PEG is used to describe certain embodiments of this invention.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods known to those of ordinary skill in the art(Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3,pages 138-161). The term “PEG” is used broadly to encompass anypolyethylene glycol molecule, without regard to size or to modificationat an end of the PEG, and can be represented as linked to the FGF-21polypeptide by the formula:

XO—(CH₂CH₂O)_(n)—CH₂CH₂—Y

where n is 2 to 10,000 and X is H or a terminal modification, includingbut not limited to, a C₁₋₄ alkyl, a protecting group, or a terminalfunctional group.

In some cases, a PEG used in the invention terminates on one end withhydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). Alternatively,the PEG can terminate with a reactive group, thereby forming abifunctional polymer. Typical reactive groups can include those reactivegroups that are commonly used to react with the functional groups foundin the 20 common amino acids (including but not limited to, maleimidegroups, activated carbonates (including but not limited to,p-nitrophenyl ester), activated esters (including but not limited to,N-hydroxysuccinimide, p-nitrophenyl ester) and aldehydes) as well asfunctional groups that are inert to the 20 common amino acids but thatreact specifically with complementary functional groups present innon-naturally encoded amino acids (including but not limited to, azidegroups, alkyne groups). It is noted that the other end of the PEG, whichis shown in the above formula by Y, will attach either directly orindirectly to a FGF-21 polypeptide via a naturally-occurring ornon-naturally encoded amino acid. For instance, Y may be an amide,carbamate or urea linkage to an amine group (including but not limitedto, the epsilon amine of lysine or the N-terminus) of the polypeptide.Alternatively, Y may be a maleimide linkage to a thiol group (includingbut not limited to, the thiol group of cysteine). Alternatively, Y maybe a linkage to a residue not commonly accessible via the 20 commonamino acids. For example, an azide group on the PEG can be reacted withan alkyne group on the FGF-21 polypeptide to form a Huisgen [3+2]cycloaddition product. Alternatively, an alkyne group on the PEG can bereacted with an azide group present in a non-naturally encoded aminoacid to form a similar product. In some embodiments, a strongnucleophile (including but not limited to, hydrazine, hydrazide,hydroxylamine, semicarbazide) can be reacted with an aldehyde or ketonegroup present in a non-naturally encoded amino acid to form a hydrazone,oxime or semicarbazone, as applicable, which in some cases can befurther reduced by treatment with an appropriate reducing agent.Alternatively, the strong nucleophile can be incorporated into theFGF-21 polypeptide via a non-naturally encoded amino acid and used toreact preferentially with a ketone or aldehyde group present in thewater soluble polymer.

Any molecular mass for a PEG can be used as practically desired,including but not limited to, from about 100 Daltons (Da) to 100,000 Daor more as desired (including but not limited to, sometimes 0.1-50 kDaor 10-40 kDa). The molecular weight of PEG may be of a wide range,including but not limited to, between about 100 Da and about 100,000 Daor more. PEG may be between about 100 Da and about 100,000 Da, includingbut not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da,45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, PEG isbetween about 100 Da and about 50,000 Da. In some embodiments, PEG isbetween about 100 Da and about 40,000 Da. In some embodiments, PEG isbetween about 1,000 Da and about 40,000 Da. In some embodiments, PEG isbetween about 5,000 Da and about 40,000 Da. In some embodiments, PEG isbetween about 10,000 Da and about 40,000 Da. Branched chain PEGs,including but not limited to, PEG molecules with each chain having a MWranging from 1-100 kDa (including but not limited to, 1-50 kDa or 5-20kDa) can also be used. The molecular weight of each chain of thebranched chain PEG may be, including but not limited to, between about1,000 Da and about 100,000 Da or more. The molecular weight of eachchain of the branched chain PEG may be between about 1,000 Da and about100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da,55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. In someembodiments, the molecular weight of each chain of the branched chainPEG is between about 1,000 Da and about 50,000 Da. In some embodiments,the molecular weight of each chain of the branched chain PEG is betweenabout 1,000 Da and about 40,000 Da. In some embodiments, the molecularweight of each chain of the branched chain PEG is between about 5,000 Daand about 40,000 Da. In some embodiments, the molecular weight of eachchain of the branched chain PEG is between about 5,000 Da and about20,000 Da. A wide range of PEG molecules are described in, including butnot limited to, the Shearwater Polymers, Inc. catalog, NektarTherapeutics catalog, incorporated herein by reference.

Generally, at least one terminus of the PEG molecule is available forreaction with the non-naturally-encoded amino acid. For example, PEGderivatives bearing alkyne and azide moieties for reaction with aminoacid side chains can be used to attach PEG to non-naturally encodedamino acids as described herein. If the non-naturally encoded amino acidcomprises an azide, then the PEG will typically contain either an alkynemoiety to effect formation of the [3+2] cycloaddition product or anactivated PEG species (i.e., ester, carbonate) containing a phosphinegroup to effect formation of the amide linkage. Alternatively, if thenon-naturally encoded amino acid comprises an alkyne, then the PEG willtypically contain an azide moiety to effect formation of the [3+2]Huisgen cycloaddition product. If the non-naturally encoded amino acidcomprises a carbonyl group, the PEG will typically comprise a potentnucleophile (including but not limited to, a hydrazide, hydrazine,hydroxylamine, or semicarbazide functionality) in order to effectformation of corresponding hydrazone, oxime, and semicarbazone linkages,respectively. In other alternatives, a reverse of the orientation of thereactive groups described above can be used, i.e., an azide moiety inthe non-naturally encoded amino acid can be reacted with a PEGderivative containing an alkyne.

In some embodiments, the FGF-21 polypeptide variant with a PEGderivative contains a chemical functionality that is reactive with thechemical functionality present on the side chain of the non-naturallyencoded amino acid.

The invention provides in some embodiments azide- andacetylene-containing polymer derivatives comprising a water solublepolymer backbone having an average molecular weight from about 800 Da toabout 100,000 Da. The polymer backbone of the water-soluble polymer canbe poly(ethylene glycol). However, it should be understood that a widevariety of water soluble polymers including but not limited topoly(ethylene)glycol and other related polymers, including poly(dextran)and poly(propylene glycol), are also suitable for use in the practice ofthis invention and that the use of the term PEG or poly(ethylene glycol)is intended to encompass and include all such molecules. The term PEGincludes, but is not limited to, poly(ethylene glycol) in any of itsforms, including bifunctional PEG, multiarmed PEG, derivatized PEG,forked PEG, branched PEG, pendent PEG (i.e. PEG or related polymershaving one or more functional groups pendent to the polymer backbone),or PEG with degradable linkages therein.

PEG is typically clear, colorless, odorless, soluble in water, stable toheat, inert to many chemical agents, does not hydrolyze or deteriorate,and is generally non-toxic. Poly(ethylene glycol) is considered to bebiocompatible, which is to say that PEG is capable of coexistence withliving tissues or organisms without causing harm. More specifically, PEGis substantially non-immunogenic, which is to say that PEG does not tendto produce an immune response in the body. When attached to a moleculehaving some desirable function in the body, such as a biologicallyactive agent, the PEG tends to mask the agent and can reduce oreliminate any immune response so that an organism can tolerate thepresence of the agent. PEG conjugates tend not to produce a substantialimmune response or cause clotting or other undesirable effects. PEGhaving the formula CH₂CH₂O—(CH₂CH₂O)_(n) CH₂CH₂—, where n is from about3 to about 4000, typically from about 20 to about 2000, is suitable foruse in the present invention. PEG having a molecular weight of fromabout 800 Da to about 100,000 Da are in some embodiments of the presentinvention particularly useful as the polymer backbone. The molecularweight of PEG may be of a wide range, including but not limited to,between about 100 Da and about 100,000 Da or more. The molecular weightof PEG may be between about 100 Da and about 100,000 Da, including butnot limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da,75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da,10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da,3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da,400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecularweight of PEG is between about 100 Da and about 50,000 Da. In someembodiments, the molecular weight of PEG is between about 100 Da andabout 40,000 Da. In some embodiments, the molecular weight of PEG isbetween about 1,000 Da and about 40,000 Da. In some embodiments, themolecular weight of PEG is between about 5,000 Da and about 40,000 Da.In some embodiments, the molecular weight of PEG is between about 10,000Da and about 40,000 Da.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, glycerol oligomers, pentaerythritoland sorbitol. The central branch moiety can also be derived from severalamino acids, such as lysine. The branched poly(ethylene glycol) can berepresented in general form as R(-PEG-OH)_(m) in which R is derived froma core moiety, such as glycerol, glycerol oligomers, or pentaerythritol,and m represents the number of arms. Multi-armed PEG molecules, such asthose described in U.S. Pat. Nos. 5,932,462; 5,643,575; 5,229,490;4,289,872; U.S. Pat. Appl. 2003/0143596; WO 96/21469; and WO 93/21259,each of which is incorporated by reference herein in its entirety, canalso be used as the polymer backbone.

Branched PEG can also be in the form of a forked PEG represented byPEG(—YCHZ2)_(n), where Y is a linking group and Z is an activatedterminal group linked to CH by a chain of atoms of defined length.

Yet another branched form, the pendant PEG, has reactive groups, such ascarboxyl, along the PEG backbone rather than at the end of PEG chains.

In addition to these forms of PEG, the polymer can also be prepared withweak or degradable linkages in the backbone. For example, PEG can beprepared with ester linkages in the polymer backbone that are subject tohydrolysis. As shown below, this hydrolysis results in cleavage of thepolymer into fragments of lower molecular weight:

-PEG-CO₂-PEG-+H₂O→PEG-CO₂H+HO-PEG-

It is understood by those of ordinary skill in the art that the termpoly(ethylene glycol) or PEG represents or includes all the forms knownin the art including but not limited to those disclosed herein.

Many other polymers are also suitable for use in the present invention.In some embodiments, polymer backbones that are water-soluble, with from2 to about 300 termini, are particularly useful in the invention.Examples of suitable polymers include, but are not limited to, otherpoly(alkylene glycols), such as poly(propylene glycol) (“PPG”),copolymers thereof (including but not limited to copolymers of ethyleneglycol and propylene glycol), terpolymers thereof, mixtures thereof, andthe like. Although the molecular weight of each chain of the polymerbackbone can vary, it is typically in the range of from about 800 Da toabout 100,000 Da, often from about 6,000 Da to about 80,000 Da. Themolecular weight of each chain of the polymer backbone may be betweenabout 100 Da and about 100,000 Da, including but not limited to, 100,000Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da,65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da,8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da,1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200Da, and 100 Da. In some embodiments, the molecular weight of each chainof the polymer backbone is between about 100 Da and about 50,000 Da. Insome embodiments, the molecular weight of each chain of the polymerbackbone is between about 100 Da and about 40,000 Da. In someembodiments, the molecular weight of each chain of the polymer backboneis between about 1,000 Da and about 40,000 Da. In some embodiments, themolecular weight of each chain of the polymer backbone is between about5,000 Da and about 40,000 Da. In some embodiments, the molecular weightof each chain of the polymer backbone is between about 10,000 Da andabout 40,000 Da.

Those of ordinary skill in the art will recognize that the foregoinglist for substantially water soluble backbones is by no means exhaustiveand is merely illustrative, and that all polymeric materials having thequalities described above are contemplated as being suitable for use inthe present invention.

In some embodiments of the present invention the polymer derivatives are“multi-functional”, meaning that the polymer backbone has at least twotermini, and possibly as many as about 300 termini, functionalized oractivated with a functional group. Multifunctional polymer derivativesinclude, but are not limited to, linear polymers having two termini,each terminus being bonded to a functional group which may be the sameor different.

In one embodiment, the polymer derivative has the structure:

X-A-POLY-B—N═N═N

wherein:N═N═N is an azide moiety;B is a linking moiety, which may be present or absent;POLY is a water-soluble non-antigenic polymer;A is a linking moiety, which may be present or absent and which may bethe same as B or different; andX is a second functional group.

Examples of a linking moiety for A and B include, but are not limitedto, a multiply-functionalized alkyl group containing up to 18, and maycontain between 1-10 carbon atoms. A heteroatom such as nitrogen, oxygenor sulfur may be included with the alkyl chain. The alkyl chain may alsobe branched at a heteroatom. Other examples of a linking moiety for Aand B include, but are not limited to, a multiply functionalized arylgroup, containing up to 10 and may contain 5-6 carbon atoms. The arylgroup may be substituted with one more carbon atoms, nitrogen, oxygen orsulfur atoms. Other examples of suitable linking groups include thoselinking groups described in U.S. Pat. Nos. 5,932,462; 5,643,575; andU.S. Pat. Appl. Publication 2003/0143596, each of which is incorporatedby reference herein. Those of ordinary skill in the art will recognizethat the foregoing list for linking moieties is by no means exhaustiveand is merely illustrative, and that all linking moieties having thequalities described above are contemplated to be suitable for use in thepresent invention.

Examples of suitable functional groups for use as X include, but are notlimited to, hydroxyl, protected hydroxyl, alkoxyl, active ester, such asN-hydroxysuccinimidyl esters and 1-benzotriazolyl esters, activecarbonate, such as N-hydroxysuccinimidyl carbonates and 1-benzotriazolylcarbonates, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate,methacrylate, acrylamide, active sulfone, amine, aminooxy, protectedamine, hydrazide, protected hydrazide, protected thiol, carboxylic acid,protected carboxylic acid, isocyanate, isothiocyanate, maleimide,vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide,glyoxals, diones, mesylates, tosylates, tresylate, alkene, ketone, andazide. As is understood by those of ordinary skill in the art, theselected X moiety should be compatible with the azide group so thatreaction with the azide group does not occur. The azide-containingpolymer derivatives may be homobifunctional, meaning that the secondfunctional group (i.e., X) is also an azide moiety, orheterobifunctional, meaning that the second functional group is adifferent functional group.

The term “protected” refers to the presence of a protecting group ormoiety that prevents reaction of the chemically reactive functionalgroup under certain reaction conditions. The protecting group will varydepending on the type of chemically reactive group being protected. Forexample, if the chemically reactive group is an amine or a hydrazide,the protecting group can be selected from the group oftert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). Ifthe chemically reactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also be usedin the present invention.

Specific examples of terminal functional groups in the literatureinclude, but are not limited to, N-succinimidyl carbonate (see e.g.,U.S. Pat. Nos. 5,281,698, 5,468,478), amine (see, e.g., Buckmann et al.Makromol. Chem. 182:1379 (1981), Zalipsky et al. Eur. Polym. J. 19:1177(1983)), hydrazide (See, e.g., Andresz et al. Makromol. Chem. 179:301(1978)), succinimidyl propionate and succinimidyl butanoate (see, e.g.,Olson et al. in Poly(ethylene glycol) Chemistry & BiologicalApplications, pp 170-181, Harris & Zalipsky Eds., ACS, Washington, D.C.,1997; see also U.S. Pat. No. 5,672,662), succinimidyl succinate (See,e.g., Abuchowski et al. Cancer Biochem. Biophys. 7:175 (1984) andJoppich et al. Makromol. Chem. 180:1381 (1979), succinimidyl ester (see,e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonate (see, e.g., U.S.Pat. No. 5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. JBiochem. 94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354(1991), oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal.Biochem. 131:25 (1983), Tondelli et al. J. Controlled Release 1:251(1985)), p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl.Biochem. Biotech., 11: 141 (1985); and Sartore et al., Appl. Biochem.Biotech., 27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym.Sci. Chem. Ed. 22:341 (1984), U.S. Pat. Nos. 5,824,784, 5,252,714),maleimide (see, e.g., Goodson et al. Biotechnology (NY) 8:343 (1990),Romani et al. in Chemistry of Peptides and Proteins 2:29 (1984)), andKogan, Synthetic Comm. 22:2417 (1992)), orthopyridyl-disulfide (see,e.g., Woghiren, et al. Bioconj. Chem. 4:314(1993)), acrylol (see, e.g.,Sawhney et al., Macromolecules, 26:581 (1993)), vinylsulfone (see, e.g.,U.S. Pat. No. 5,900,461). All of the above references and patents areincorporated herein by reference.

In certain embodiments of the present invention, the polymer derivativesof the invention comprise a polymer backbone having the structure:

X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—N═N═N

wherein:X is a functional group as described above; andn is about 20 to about 4000.

In another embodiment, the polymer derivatives of the invention comprisea polymer backbone having the structure:

X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂O—(CH₂)_(m)—W—N═N═N

wherein:W is an aliphatic or aromatic linker moiety comprising between 1-10carbon atoms;n is about 20 to about 4000; andX is a functional group as described above. m is between 1 and 10.

The azide-containing PEG derivatives of the invention can be prepared bya variety of methods known in the art and/or disclosed herein. In onemethod, shown below, a water soluble polymer backbone having an averagemolecular weight from about 800 Da to about 100,000 Da, the polymerbackbone having a first terminus bonded to a first functional group anda second terminus bonded to a suitable leaving group, is reacted with anazide anion (which may be paired with any of a number of suitablecounter-ions, including sodium, potassium, tert-butylammonium and soforth). The leaving group undergoes a nucleophilic displacement and isreplaced by the azide moiety, affording the desired azide-containing PEGpolymer.

X-PEG-L+N₃ ⁻→X-PEG-N₃

As shown, a suitable polymer backbone for use in the present inventionhas the formula X-PEG-L, wherein PEG is poly(ethylene glycol) and X is afunctional group which does not react with azide groups and L is asuitable leaving group. Examples of suitable functional groups include,but are not limited to, hydroxyl, protected hydroxyl, acetal, alkenyl,amine, aminooxy, protected amine, protected hydrazide, protected thiol,carboxylic acid, protected carboxylic acid, maleimide, dithiopyridine,and vinylpyridine, and ketone. Examples of suitable leaving groupsinclude, but are not limited to, chloride, bromide, iodide, mesylate,tresylate, and tosylate.

In another method for preparation of the azide-containing polymerderivatives of the present invention, a linking agent bearing an azidefunctionality is contacted with a water soluble polymer backbone havingan average molecular weight from about 800 Da to about 100,000 Da,wherein the linking agent bears a chemical functionality that will reactselectively with a chemical functionality on the PEG polymer, to form anazide-containing polymer derivative product wherein the azide isseparated from the polymer backbone by a linking group.

An exemplary reaction scheme is shown below:

X-PEG-M+N-linker-N═N═N→PG-X-PEG-linker-N═N═N

wherein:PEG is poly(ethylene glycol) and X is a capping group such as alkoxy ora functional group as described above; andM is a functional group that is not reactive with the azidefunctionality but that will react efficiently and selectively with the Nfunctional group.

Examples of suitable functional groups include, but are not limited to,M being a carboxylic acid, carbonate or active ester if N is an amine; Mbeing a ketone if N is a hydrazide or aminooxy moiety; M being a leavinggroup if N is a nucleophile.

Purification of the crude product may be accomplished by known methodsincluding, but are not limited to, precipitation of the product followedby chromatography, if necessary.

A more specific example is shown below in the case of PEG diamine, inwhich one of the amines is protected by a protecting group moiety suchas tert-butyl-Boc and the resulting mono-protected PEG diamine isreacted with a linking moiety that bears the azide functionality:

BocHN-PEG-NH₂+HO₂C—(CH₂)₃—N═N═N

In this instance, the amine group can be coupled to the carboxylic acidgroup using a variety of activating agents such as thionyl chloride orcarbodiimide reagents and N-hydroxysuccinimide or N-hydroxybenzotriazoleto create an amide bond between the monoamine PEG derivative and theazide-bearing linker moiety. After successful formation of the amidebond, the resulting N-tert-butyl-Boc-protected azide-containingderivative can be used directly to modify bioactive molecules or it canbe further elaborated to install other useful functional groups. Forinstance, the N-t-Boc group can be hydrolyzed by treatment with strongacid to generate an omega-amino-PEG-azide. The resulting amine can beused as a synthetic handle to install other useful functionality such asmaleimide groups, activated disulfides, activated esters and so forthfor the creation of valuable heterobifunctional reagents.

Heterobifunctional derivatives are particularly useful when it isdesired to attach different molecules to each terminus of the polymer.For example, the omega-N-amino-N-azido PEG would allow the attachment ofa molecule having an activated electrophilic group, such as an aldehyde,ketone, activated ester, activated carbonate and so forth, to oneterminus of the PEG and a molecule having an acetylene group to theother terminus of the PEG.

In another embodiment of the invention, the polymer derivative has thestructure:

X—A-POLY-B—C≡C—R

wherein:R can be either H or an alkyl, alkene, alkyoxy, or aryl or substitutedaryl group;B is a linking moiety, which may be present or absent;POLY is a water-soluble non-antigenic polymer;A is a linking moiety, which may be present or absent and which may bethe same as B or different; andX is a second functional group.

Examples of a linking moiety for A and B include, but are not limitedto, a multiply-functionalized alkyl group containing up to 18, and maycontain between 1-10 carbon atoms. A heteroatom such as nitrogen, oxygenor sulfur may be included with the alkyl chain. The alkyl chain may alsobe branched at a heteroatom. Other examples of a linking moiety for Aand B include, but are not limited to, a multiply functionalized arylgroup, containing up to 10 and may contain 5-6 carbon atoms. The arylgroup may be substituted with one more carbon atoms, nitrogen, oxygen,or sulfur atoms. Other examples of suitable linking groups include thoselinking groups described in U.S. Pat. Nos. 5,932,462 and 5,643,575 andU.S. Pat. Appl. Publication 2003/0143596, each of which is incorporatedby reference herein. Those of ordinary skill in the art will recognizethat the foregoing list for linking moieties is by no means exhaustiveand is intended to be merely illustrative, and that a wide variety oflinking moieties having the qualities described above are contemplatedto be useful in the present invention.

Examples of suitable functional groups for use as X include hydroxyl,protected hydroxyl, alkoxyl, active ester, such as N-hydroxysuccinimidylesters and 1-benzotriazolyl esters, active carbonate, such asN-hydroxysuccinimidyl carbonates and 1-benzotriazolyl carbonates,acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide,protected hydrazide, protected thiol, carboxylic acid, protectedcarboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones,mesylates, tosylates, and tresylate, alkene, ketone, and acetylene. Aswould be understood, the selected X moiety should be compatible with theacetylene group so that reaction with the acetylene group does notoccur. The acetylene-containing polymer derivatives may behomobifunctional, meaning that the second functional group (i.e., X) isalso an acetylene moiety, or heterobifunctional, meaning that the secondfunctional group is a different functional group.

In another embodiment of the present invention, the polymer derivativescomprise a polymer backbone having the structure:

X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—O—(CH₂)_(m)—C≡CH

wherein:X is a functional group as described above;n is about 20 to about 4000; andm is between 1 and 10.Specific examples of each of the heterobifunctional PEG polymers areshown below.

The acetylene-containing PEG derivatives of the invention can beprepared using methods known to those of ordinary skill in the artand/or disclosed herein. In one method, a water soluble polymer backbonehaving an average molecular weight from about 800 Da to about 100,000Da, the polymer backbone having a first terminus bonded to a firstfunctional group and a second terminus bonded to a suitable nucleophilicgroup, is reacted with a compound that bears both an acetylenefunctionality and a leaving group that is suitable for reaction with thenucleophilic group on the PEG. When the PEG polymer bearing thenucleophilic moiety and the molecule bearing the leaving group arecombined, the leaving group undergoes a nucleophilic displacement and isreplaced by the nucleophilic moiety, affording the desiredacetylene-containing polymer.

X-PEG-Nu+L-A-C→X-PEG-Nu-A-C≡CR′

As shown, a preferred polymer backbone for use in the reaction has theformula X-PEG-Nu, wherein PEG is poly(ethylene glycol), Nu is anucleophilic moiety and X is a functional group that does not react withNu, L or the acetylene functionality.

Examples of Nu include, but are not limited to, amine, alkoxy, aryloxy,sulfhydryl, imino, carboxylate, hydrazide, aminoxy groups that wouldreact primarily via a SN2-type mechanism. Additional examples of Nugroups include those functional groups that would react primarily via annucleophilic addition reaction. Examples of L groups include chloride,bromide, iodide, mesylate, tresylate, and tosylate and other groupsexpected to undergo nucleophilic displacement as well as ketones,aldehydes, thioesters, olefins, alpha-beta unsaturated carbonyl groups,carbonates and other electrophilic groups expected to undergo additionby nucleophiles.

In another embodiment of the present invention, A is an aliphatic linkerof between 1-10 carbon atoms or a substituted aryl ring of between 6-14carbon atoms. X is a functional group which does not react with azidegroups and L is a suitable leaving group

In another method for preparation of the acetylene-containing polymerderivatives of the invention, a PEG polymer having an average molecularweight from about 800 Da to about 100,000 Da, bearing either a protectedfunctional group or a capping agent at one terminus and a suitableleaving group at the other terminus is contacted by an acetylene anion.

An exemplary reaction scheme is shown below:

X-PEG-L+—C≡CR′→X-PEG-C≡CR′

wherein:PEG is poly(ethylene glycol) and X is a capping group such as alkoxy ora functional group as described above; andR′ is either H, an alkyl, alkoxy, aryl or aryloxy group or a substitutedalkyl, alkoxyl, aryl or aryloxy group.

In the example above, the leaving group L should be sufficientlyreactive to undergo SN2-type displacement when contacted with asufficient concentration of the acetylene anion. The reaction conditionsrequired to accomplish SN2 displacement of leaving groups by acetyleneanions are known to those of ordinary skill in the art.

Purification of the crude product can usually be accomplished by methodsknown in the art including, but are not limited to, precipitation of theproduct followed by chromatography, if necessary.

Water soluble polymers can be linked to the FGF-21 polypeptides of theinvention. The water soluble polymers may be linked via a non-naturallyencoded amino acid incorporated in the FGF-21 polypeptide or anyfunctional group or substituent of a non-naturally encoded or naturallyencoded amino acid, or any functional group or substituent added to anon-naturally encoded or naturally encoded amino acid. Alternatively,the water soluble polymers are linked to a FGF-21 polypeptideincorporating a non-naturally encoded amino acid via anaturally-occurring amino acid (including but not limited to, cysteine,lysine or the amine group of the N-terminal residue). In some cases, theFGF-21 polypeptides of the invention comprise 1, 2, 3, 4, 5, 6, 7, 8, 9,10 non-natural amino acids, wherein one or more non-naturally-encodedamino acid(s) are linked to water soluble polymer(s) (including but notlimited to, PEG and/or oligosaccharides). In some cases, the FGF-21polypeptides of the invention further comprise 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more naturally-encoded amino acid(s) linked to water solublepolymers. In some cases, the FGF-21 polypeptides of the inventioncomprise one or more non-naturally encoded amino acid(s) linked to watersoluble polymers and one or more naturally-occurring amino acids linkedto water soluble polymers. In some embodiments, the water solublepolymers used in the present invention enhance the serum half-life ofthe FGF-21 polypeptide relative to the unconjugated form.

The number of water soluble polymers linked to a FGF-21 polypeptide(i.e., the extent of PEGylation or glycosylation) of the presentinvention can be adjusted to provide an altered (including but notlimited to, increased or decreased) pharmacologic, pharmacokinetic orpharmacodynamic characteristic such as in vivo half-life. In someembodiments, the half-life of FGF-21 is increased at least about 10, 20,30, 40, 50, 60, 70, 80, 90 percent, 2-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold,16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold,40-fold, 50-fold, or at least about 100-fold over an unmodifiedpolypeptide.

PEG derivatives containing a strong nucleophilic group (i.e., hydrazide,hydrazine, hydroxylamine or semicarbazide)

In one embodiment of the present invention, a FGF-21 polypeptidecomprising a carbonyl-containing non-naturally encoded amino acid ismodified with a PEG derivative that contains a terminal hydrazine,hydroxylamine, hydrazide or semicarbazide moiety that is linked directlyto the PEG backbone.

In some embodiments, the hydroxylamine-terminal PEG derivative will havethe structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—O—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In some embodiments, the hydrazine- or hydrazide-containing PEGderivative will have the structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—X—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 and X is optionally a carbonyl group (C═O) that can bepresent or absent.

In some embodiments, the semicarbazide-containing PEG derivative willhave the structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—NH—C(O)—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000.

In another embodiment of the invention, a FGF-21 polypeptide comprisinga carbonyl-containing amino acid is modified with a PEG derivative thatcontains a terminal hydroxylamine, hydrazide, hydrazine, orsemicarbazide moiety that is linked to the PEG backbone by means of anamide linkage.

In some embodiments, the hydroxylamine-terminal PEG derivatives have thestructure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—O—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In some embodiments, the hydrazine- or hydrazide-containing PEGderivatives have the structure:

RO—(CH₂CH₂O)_(n)-O—(CH₂)₂—NH—C(O)(CH₂)_(m)—X—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, n is100-1,000 and X is optionally a carbonyl group (C═O) that can be presentor absent.

In some embodiments, the semicarbazide-containing PEG derivatives havethe structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—NH—C(O)—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000.

In another embodiment of the invention, a FGF-21 polypeptide comprisinga carbonyl-containing amino acid is modified with a branched PEGderivative that contains a terminal hydrazine, hydroxylamine, hydrazideor semicarbazide moiety, with each chain of the branched PEG having a MWranging from 10-40 kDa and, may be from 5-20 kDa.

In another embodiment of the invention, a FGF-21 polypeptide comprisinga non-naturally encoded amino acid is modified with a PEG derivativehaving a branched structure. For instance, in some embodiments, thehydrazine- or hydrazide-terminal PEG derivative will have the followingstructure:

[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)]₂CH(CH₂)_(m)—X—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000, and X is optionally a carbonyl group (C═O) that can bepresent or absent.

In some embodiments, the PEG derivatives containing a semicarbazidegroup will have the structure:

[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—C(O)—NH—CH₂—CH₂]₂CH—X—(CH₂)_(m)—NH—C(O)—NH—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionallyNH, O, S, C(O) or not present, m is 2-10 and n is 100-1,000.

In some embodiments, the PEG derivatives containing a hydroxylaminegroup will have the structure:

[RO—(CH₂CH₂O)_(n)-O—(CH₂)₂—C(O)—NH—CH₂—CH_(2]2)CH—X—(CH₂)_(m)—O—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionallyNH, O, S, C(O) or not present, m is 2-10 and n is 100-1,000.

The degree and sites at which the water soluble polymer(s) are linked tothe FGF-21 polypeptide can modulate the binding of the FGF-21polypeptide to the FGF-21 polypeptide receptor. In some embodiments, thelinkages are arranged such that the FGF-21 polypeptide binds the FGF-21polypeptide receptor with a K_(d) of about 400 nM or lower, with a K_(d)of 150 nM or lower, and in some cases with a K_(d) of 100 nM or lower,as measured by an equilibrium binding assay, such as that described inSpencer et al., J. Biol. Chem., 263:7862-7867 (1988) for FGF-21.

Methods and chemistry for activation of polymers as well as forconjugation of peptides are described in the literature and are known inthe art. Commonly used methods for activation of polymers include, butare not limited to, activation of functional groups with cyanogenbromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin,divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine, etc.(see, R. F. Taylor, (1991), PROTEIN IMMOBILISATION. FUNDAMENTAL ANDAPPLICATIONS, Marcel Dekker, N.Y.; S. S. Wong, (1992), CHEMISTRY OFPROTEIN CONJUGATION AND CROSSLINKING, CRC Press, Boca Raton; G. T.Hermanson et al., (1993), IMMOBILIZED AFFINITY LIGAND TECHNIQUES,Academic Press, N.Y.; Dunn, R. L., et al., Eds. POLYMERIC DRUGS AND DRUGDELIVERY SYSTEMS, ACS Symposium Series Vol. 469, American ChemicalSociety, Washington, D.C. 1991).

Several reviews and monographs on the functionalization and conjugationof PEG are available. See, for example, Harris, Macromol. Chem. Phys.C25: 325-373 (1985); Scouten, Methods in Enzymology 135: 30-65 (1987);Wong et al., Enzyme Microb. Technol. 14: 866-874 (1992); Delgado et al.,Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992);Zalipsky, Bioconjugate Chem. 6: 150-165 (1995).

Methods for activation of polymers can also be found in WO 94/17039,U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. Nos.5,219,564, 5,122,614, WO 90/13540, U.S. Pat. No. 5,281,698, and WO93/15189, and for conjugation between activated polymers and enzymesincluding but not limited to Coagulation Factor VIII (WO 94/15625),hemoglobin (WO 94/09027), oxygen carrying molecule (U.S. Pat. No.4,412,989), ribonuclease and superoxide dismutase (Veronese at al., App.Biochem. Biotech. 11: 141-52 (1985)). All references and patents citedare incorporated by reference herein.

PEGylation (i.e., addition of any water soluble polymer) of FGF-21polypeptides containing a non-naturally encoded amino acid, such asp-azido-L-phenylalanine, is carried out by any convenient method. Forexample, FGF-21 polypeptide is PEGylated with an alkyne-terminated mPEGderivative. Briefly, an excess of solid mPEG(5000)-O—CH₂—C≡CH is added,with stirring, to an aqueous solution of p-azido-L-Phe-containing FGF-21polypeptide at room temperature. Typically, the aqueous solution isbuffered with a buffer having a pK_(a) near the pH at which the reactionis to be carried out (generally about pH 4-10). Examples of suitablebuffers for PEGylation at pH 7.5, for instance, include, but are notlimited to, HEPES, phosphate, borate, TRIS-HCl, EPPS, and TES. The pH iscontinuously monitored and adjusted if necessary. The reaction istypically allowed to continue for between about 1-48 hours.

The reaction products are subsequently subjected to hydrophobicinteraction chromatography to separate the PEGylated FGF-21 polypeptidevariants from free mPEG(5000)-O—CH₂—C≡CH and any high-molecular weightcomplexes of the pegylated FGF-21 polypeptide which may form whenunblocked PEG is activated at both ends of the molecule, therebycrosslinking FGF-21 polypeptide variant molecules. The conditions duringhydrophobic interaction chromatography are such that freemPEG(5000)-O—CH₂—C≡CH flows through the column, while any crosslinkedPEGylated FGF-21 polypeptide variant complexes elute after the desiredforms, which contain one FGF-21 polypeptide variant molecule conjugatedto one or more PEG groups. Suitable conditions vary depending on therelative sizes of the cross-linked complexes versus the desiredconjugates and are readily determined by those of ordinary skill in theart. The eluent containing the desired conjugates is concentrated byultrafiltration and desalted by diafiltration.

If necessary, the PEGylated FGF-21 polypeptide obtained from thehydrophobic chromatography can be purified further by one or moreprocedures known to those of ordinary skill in the art including, butare not limited to, affinity chromatography; anion- or cation-exchangechromatography (using, including but not limited to, DEAE SEPHAROSE);chromatography on silica; reverse phase HPLC; gel filtration (using,including but not limited to, SEPHADEX G-75); hydrophobic interactionchromatography; size-exclusion chromatography, metal-chelatechromatography; ultrafiltration/diafiltration; ethanol precipitation;ammonium sulfate precipitation; chromatofocusing; displacementchromatography; electrophoretic procedures (including but not limited topreparative isoelectric focusing), differential solubility (includingbut not limited to ammonium sulfate precipitation), or extraction.Apparent molecular weight may be estimated by GPC by comparison toglobular protein standards (Preneta, AZ in PROTEIN PURIFICATION METHODS,A PRACTICAL APPROACH (Harris & Angal, Eds.) IRL Press 1989, 293-306).The purity of the FGF-21-PEG conjugate can be assessed by proteolyticdegradation (including but not limited to, trypsin cleavage) followed bymass spectrometry analysis. Pepinsky R B., et al., J. Pharmcol. & Exp.Ther. 297(3):1059-66 (2001).

A water soluble polymer linked to an amino acid of a FGF-21 polypeptideof the invention can be further derivatized or substituted withoutlimitation.

Azide-containing PEG derivatives

In another embodiment of the invention, a FGF-21 polypeptide is modifiedwith a PEG derivative that contains an azide moiety that will react withan alkyne moiety present on the side chain of the non-naturally encodedamino acid. In general, the PEG derivatives will have an averagemolecular weight ranging from 1-100 kDa and, in some embodiments, from10-40 kDa.

In some embodiments, the azide-terminal PEG derivative will have thestructure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—N₃

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In another embodiment, the azide-terminal PEG derivative will have thestructure:

RO—(CH₂CH₂O)_(n)-O—(CH₂)_(m)—NH—C(O)—(CH₂)_(p)—N₃

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10 and n is 100-1,000 (i.e., average molecular weight is between 5-40kDa).

In another embodiment of the invention, a FGF-21 polypeptide comprisinga alkyne-containing amino acid is modified with a branched PEGderivative that contains a terminal azide moiety, with each chain of thebranched PEG having a MW ranging from 10-40 kDa and may be from 5-20kDa. For instance, in some embodiments, the azide-terminal PEGderivative will have the following structure:

[RO—(CH₂CH₂O)_(n)-O—(CH₂)₂—NH—C(O)]₂CH(CH₂)_(m)—X—(CH₂)_(p)N₃

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10, and n is 100-1,000, and X is optionally an O, N, S or carbonylgroup (C═O), in each case that can be present or absent.Alkyne-containing PEG derivatives

In another embodiment of the invention, a FGF-21 polypeptide is modifiedwith a PEG derivative that contains an alkyne moiety that will reactwith an azide moiety present on the side chain of the non-naturallyencoded amino acid.

In some embodiments, the alkyne-terminal PEG derivative will have thefollowing structure:

RO—(CH₂CH₂O)_(n)-O—(CH₂)_(m)—C≡CH

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In another embodiment of the invention, a FGF-21 polypeptide comprisingan alkyne-containing non-naturally encoded amino acid is modified with aPEG derivative that contains a terminal azide or terminal alkyne moietythat is linked to the PEG backbone by means of an amide linkage.

In some embodiments, the alkyne-terminal PEG derivative will have thefollowing structure:

RO—(CH₂CH₂O)_(n)-O—(CH₂)_(m)—NH—C(O)—(CH₂)_(p)—C≡CH

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10 and n is 100-1,000.

In another embodiment of the invention, a FGF-21 polypeptide comprisingan azide-containing amino acid is modified with a branched PEGderivative that contains a terminal alkyne moiety, with each chain ofthe branched PEG having a MW ranging from 10-40 kDa and may be from 5-20kDa. For instance, in some embodiments, the alkyne-terminal PEGderivative will have the following structure:

[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)]₂CH(CH₂)_(m)—X—(CH₂)_(p) C≡CH

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10, and n is 100-1,000, and X is optionally an O, N, S or carbonylgroup (C═O), or not present.Phosphine-containing PEG derivatives

In another embodiment of the invention, a FGF-21 polypeptide is modifiedwith a PEG derivative that contains an activated functional group(including but not limited to, ester, carbonate) further comprising anaryl phosphine group that will react with an azide moiety present on theside chain of the non-naturally encoded amino acid. In general, the PEGderivatives will have an average molecular weight ranging from 1-100 kDaand, in some embodiments, from 10-40 kDa.

In some embodiments, the PEG derivative will have the structure:

wherein n is 1-10; X can be O, N, S or not present, Ph is phenyl, and Wis a water soluble polymer.

In some embodiments, the PEG derivative will have the structure:

wherein X can be O, N, S or not present, Ph is phenyl, W is a watersoluble polymer and R can be H, alkyl, aryl, substituted alkyl andsubstituted aryl groups. Exemplary R groups include but are not limitedto —CH₂, —C(CH₃) 3, —OR′, —NR′R″, —SR′, -halogen, —C(O)R′, —CONR′R″,—S(O)₂R′, —S(O)₂NR′R″, —CN and —NO₂. R′, R″, R′″ and R″″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R″″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF₃ and —CH₂CF₃) and acyl (including but not limited to,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).Other PEG derivatives and General PEGylation techniques

Other exemplary PEG molecules that may be linked to FGF-21 polypeptides,as well as PEGylation methods include, but are not limited to, thosedescribed in, e.g., U.S. Patent Publication No. 2004/0001838;2002/0052009; 2003/0162949; 2004/0013637; 2003/0228274; 2003/0220447;2003/0158333; 2003/0143596; 2003/0114647; 2003/0105275; 2003/0105224;2003/0023023; 2002/0156047; 2002/0099133; 2002/0086939; 2002/0082345;2002/0072573; 2002/0052430; 2002/0040076; 2002/0037949; 2002/0002250;2001/0056171; 2001/0044526; 2001/0021763; U.S. Pat. Nos. 6,646,110;5,824,778; 5,476,653; 5,219,564; 5,629,384; 5,736,625; 4,902,502;5,281,698; 5,122,614; 5,473,034; 5,516,673; 5,382,657; 6,552,167;6,610,281; 6,515,100; 6,461,603; 6,436,386; 6,214,966; 5,990,237;5,900,461; 5,739,208; 5,672,662; 5,446,090; 5,808,096; 5,612,460;5,324,844; 5,252,714; 6,420,339; 6,201,072; 6,451,346; 6,306,821;5,559,213; 5,747,646; 5,834,594; 5,849,860; 5,980,948; 6,004,573;6,129,912; WO 97/32607, EP 229,108, EP 402,378, WO 92/16555, WO94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO 94/28024, WO95/00162, WO 95/11924, WO95/13090, WO 95/33490, WO 96/00080, WO97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO 99/32139, WO99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP 439 508, WO97/03106, WO 96/21469, WO 95/13312, EP 921 131, WO 98/05363, EP 809 996,WO 96/41813, WO 96/07670, EP 605 963, EP 510 356, EP 400 472, EP 183 503and EP 154 316, which are incorporated by reference herein. Any of thePEG molecules described herein may be used in any form, including butnot limited to, single chain, branched chain, multiarm chain, singlefunctional, bi-functional, multi-functional, or any combination thereof.

Additional polymer and PEG derivatives are described in the followingpatent applications which are all incorporated by reference in theirentirety herein: U.S. Patent Publication No. 2006/0194256, U.S. PatentPublication No. 2006/0217532, U.S. Patent Publication No. 2006/0217289,U.S. Provisional Patent No. 60/755,338; U.S. Provisional Patent No.60/755,711; U.S. Provisional Patent No. 60/755,018; International PatentApplication No. PCT/US06/49397; WO 2006/069246; U.S. Provisional PatentNo. 60/743,041; U.S. Provisional Patent No. 60/743,040; InternationalPatent Application No. PCT/US06/47822; U.S. Provisional Patent No.60/882,819; U.S. Provisional Patent No. 60/882,500; and U.S. ProvisionalPatent No. 60/870,594.

Enhancing Affinity for Serum Albumin

Various molecules can also be fused to the FGF-21 polypeptides of theinvention to modulate the half-life of FGF-21 polypeptides in serum. Insome embodiments, molecules are linked or fused to FGF-21 polypeptidesof the invention to enhance affinity for endogenous serum albumin in ananimal.

For example, in some cases, a recombinant fusion of a FGF-21 polypeptideand an albumin binding sequence is made. Exemplary albumin bindingsequences include, but are not limited to, the albumin binding domainfrom streptococcal protein G (see. e.g., Makrides et al., J. Pharmacol.Exp. Ther. 277:534-542 (1996) and Sjolander et al., J. Immunol. Methods201:115-123 (1997)), or albumin-binding peptides such as those describedin, e.g., Dennis, et al., J. Biol. Chem. 277:35035-35043 (2002).

In other embodiments, the FGF-21 polypeptides of the present inventionare acylated with fatty acids. In some cases, the fatty acids promotebinding to serum albumin. See, e.g., Kurtzhals, et al., Biochem. J.312:725-731 (1995).

In other embodiments, the FGF-21 polypeptides of the invention are fuseddirectly with serum albumin (including but not limited to, human serumalbumin). Those of skill in the art will recognize that a wide varietyof other molecules can also be linked to FGF-21 in the present inventionto modulate binding to serum albumin or other serum components.

X. Glycosylation of FGF-21 Polypeptides

The invention includes FGF-21 polypeptides incorporating one or morenon-naturally encoded amino acids bearing saccharide residues. Thesaccharide residues may be either natural (including but not limited to,N-acetylglucosamine) or non-natural (including but not limited to,3-fluorogalactose). The saccharides may be linked to the non-naturallyencoded amino acids either by an N- or O-linked glycosidic linkage(including but not limited to, N-acetylgalactose-L-serine) or anon-natural linkage (including but not limited to, an oxime or thecorresponding C- or S-linked glycoside).

The saccharide (including but not limited to, glycosyl) moieties can beadded to FGF-21 polypeptides either in vivo or in vitro. In someembodiments of the invention, a FGF-21 polypeptide comprising acarbonyl-containing non-naturally encoded amino acid is modified with asaccharide derivatized with an aminooxy group to generate thecorresponding glycosylated polypeptide linked via an oxime linkage. Onceattached to the non-naturally encoded amino acid, the saccharide may befurther elaborated by treatment with glycosyltransferases and otherenzymes to generate an oligosaccharide bound to the FGF-21 polypeptide.See, e.g., H. Liu, et al. J. Am. Chem. Soc. 125: 1702-1703 (2003).

In some embodiments of the invention, a FGF-21 polypeptide comprising acarbonyl-containing non-naturally encoded amino acid is modifieddirectly with a glycan with defined structure prepared as an aminooxyderivative. One of ordinary skill in the art will recognize that otherfunctionalities, including azide, alkyne, hydrazide, hydrazine, andsemicarbazide, can be used to link the saccharide to the non-naturallyencoded amino acid.

In some embodiments of the invention, a FGF-21 polypeptide comprising anazide or alkynyl-containing non-naturally encoded amino acid can then bemodified by, including but not limited to, a Huisgen [3+2] cycloadditionreaction with, including but not limited to, alkynyl or azidederivatives, respectively. This method allows for proteins to bemodified with extremely high selectivity.

XI. FGF-21 Dimers and Multimers

The present invention also provides for FGF-21 and FGF-21 analogcombinations such as homodimers, heterodimers, homomultimers, orheteromultimers (i.e., trimers, tetramers, etc.) where FGF-21 containingone or more non-naturally encoded amino acids is bound to another FGF-21or FGF-21 variant thereof or any other polypeptide that is not FGF-21 orFGF-21 variant thereof, either directly to the polypeptide backbone orvia a linker. Due to its increased molecular weight compared tomonomers, the FGF-21 dimer or multimer conjugates may exhibit new ordesirable properties, including but not limited to differentpharmacological, pharmacokinetic, pharmacodynamic, modulated therapeutichalf-life, or modulated plasma half-life relative to the monomericFGF-21. In some embodiments, FGF-21 dimers of the invention willmodulate signal transduction of the FGF-21 receptor. In otherembodiments, the FGF-21 dimers or multimers of the present inventionwill act as a FGF-21 receptor antagonist, agonist, or modulator.

In some embodiments, one or more of the FGF-21 molecules present in aFGF-21 containing dimer or multimer comprises a non-naturally encodedamino acid linked to a water soluble polymer.

In some embodiments, the FGF-21 polypeptides are linked directly,including but not limited to, via an Asn-Lys amide linkage or Cys-Cysdisulfide linkage. In some embodiments, the FGF-21 polypeptides, and/orthe linked non-FGF-21 molecule, will comprise different non-naturallyencoded amino acids to facilitate dimerization, including but notlimited to, an alkyne in one non-naturally encoded amino acid of a firstFGF-21 polypeptide and an azide in a second non-naturally encoded aminoacid of a second molecule will be conjugated via a Huisgen [3+2]cycloaddition. Alternatively, FGF-21, and/or the linked non-FGF-21molecule comprising a ketone-containing non-naturally encoded amino acidcan be conjugated to a second polypeptide comprising ahydroxylamine-containing non-naturally encoded amino acid and thepolypeptides are reacted via formation of the corresponding oxime.

Alternatively, the two FGF-21 polypeptides, and/or the linked non-FGF-21molecule, are linked via a linker. Any hetero- or homo-bifunctionallinker can be used to link the two molecules, and/or the linkednon-FGF-21 molecules, which can have the same or different primarysequence. In some cases, the linker used to tether the FGF-21, and/orthe linked non-FGF-21 molecules together can be a bifunctional PEGreagent. The linker may have a wide range of molecular weight ormolecular length. Larger or smaller molecular weight linkers may be usedto provide a desired spatial relationship or conformation between FGF-21and the linked entity or between FGF-21 and its receptor, or between thelinked entity and its binding partner, if any. Linkers having longer orshorter molecular length may also be used to provide a desired space orflexibility between FGF-21 and the linked entity, or between the linkedentity and its binding partner, if any.

In some embodiments, the invention provides water-soluble bifunctionallinkers that have a dumbbell structure that includes: a) an azide, analkyne, a hydrazine, a hydrazide, a hydroxylamine, or acarbonyl-containing moiety on at least a first end of a polymerbackbone; and b) at least a second functional group on a second end ofthe polymer backbone. The second functional group can be the same ordifferent as the first functional group. The second functional group, insome embodiments, is not reactive with the first functional group. Theinvention provides, in some embodiments, water-soluble compounds thatcomprise at least one arm of a branched molecular structure. Forexample, the branched molecular structure can be dendritic.

In some embodiments, the invention provides multimers comprising one ormore FGF-21 polypeptide, formed by reactions with water solubleactivated polymers that have the structure:

R—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—X

wherein n is from about 5 to 3,000, m is 2-10, X can be an azide, analkyne, a hydrazine, a hydrazide, an aminooxy group, a hydroxylamine, anacetyl, or carbonyl-containing moiety, and R is a capping group, afunctional group, or a leaving group that can be the same or differentas X. R can be, for example, a functional group selected from the groupconsisting of hydroxyl, protected hydroxyl, alkoxyl,N-hydroxysuccinimidyl ester, 1-benzotriazolyl ester,N-hydroxysuccinimidyl carbonate, 1-benzotriazolyl carbonate, acetal,aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide,protected hydrazide, protected thiol, carboxylic acid, protectedcarboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones,mesylates, tosylates, and tresylate, alkene, and ketone.

XII. Measurement of FGF-21 Polypeptide Activity and Affinity of FGF-21Polypeptide for the FGF-21 Polypeptide Receptor

FGF-21 has been shown to stimulate glucose uptake and enhance insulinsensitivity in 3T₃-L1 adipocytes, an in vitro model utilized for thestudy of adipose tissue metabolism as shown in Example 3 of U.S. PatentPublication No. 20040259780 which is incorporated by reference in itsentirety. A characteristic of Type 2 diabetes is the deficiency ofglucose uptake in various tissue types including adipose tissue. Thus,FGF-21 is useful for treating Type 2 diabetes by lowering blood glucoselevels. Moreover, FGF-21 is useful for treating obesity by increasingenergy expenditure by faster and more efficient glucose utilization.Additionally, FGF-21 has been shown to stimulate glucose uptake in3T₃-L1 adipocytes in an insulin independent manner, indicating that itis useful for treating Type 1 diabetes as well. See U.S. PatentPublication No. 20040259780. FGF-21 is shown to stimulate glucose uptakein 3T₃-L1 adipocytes in a concentration dependent manner at asub-optimal concentration of insulin (5 nM) and in the absence ofinsulin in U.S. Patent Publication No. 20040259780. Additionally, FGF-21induces glucose uptake in an ex vivo tissue model, described in in U.S.Patent Publication No. 20040259780.

Glucose uptake in 3T₃-1 adipocytes may be assessed using the followingmethod. 3T₃-L1 cells are obtained from the American Type CultureCollection (ATCC, Rockville, Md.). Cells are cultured in growth medium(GM) containing 10% iron-enriched fetal bovine serum in Dulbecco'smodified Eagle's medium. For standard adipocyte differentiation, 2 daysafter cells reached confluency (referred as day 0), cells are exposed todifferentiation medium (DM) containing 10% fetal bovine serum, 10 μg/mlof insulin, 1 μM dexamethasone, and 0.5 μM isobutylmethylxanthine, for48 hours. Cells then are maintained in post differentiation mediumcontaining 10% fetal bovine serum, and 10 μg/ml of insulin. In vitropotency may be measured with the glucose uptake assay which are known tothose of ordinary skill in the art. In vitro potency can be defined asthe measure of glucose uptake of a FGF-21 compound in a cell-based assayand is a measure of the biological potency of the FGF compound. It canbe expressed as the EC₅₀ which is the effective concentration ofcompound that results in 50% activity in a single dose-responseexperiment.

Glucose Transport Assay—Insulin Dependent—Hexose uptake, as assayed bythe accumulation of 0.1 mM 2-deoxy-D-[¹⁴C]glucose, is measured asfollows: 3T₃-L1 adipocytes in 12-well plates are washed twice with KRPbuffer (136 mM NaCl, 4.7 mM KCl, 10 mM NaPO4, 0.9 mM CaCl₂, 0.9 mMMgSO₄, pH 7.4) warmed to 37° C. and containing 0.2% BSA, incubated inLeibovitz's L-15 medium containing 0.2% BSA for 2 hours at 37° C. inroom air, washed twice again with KRP containing, 0.2% BSA buffer, andincubated in KRP, 0.2% BSA buffer in the absence (Me2S0 only) orpresence of wortmannin for 30 minutes at 37° C. in room air. Insulin isthen added to a final concentration of 100 nM for 15 minutes, and theuptake of 2-deoxy-D-[¹⁴C]glucose is measured for the last 4 minutes.Nonspecific uptake, measured in the presence of 10 μM cytochalasin B, issubtracted from all values. Protein concentrations are determined withthe Pierce bicinchoninic acid assay. Uptake is measured routinely intriplicate or quadruplicate for each experiment. The effect of acute andchronic pretreatment of 3T₃-L1 adipocytes with FGF-21 in the presence ofinsulin may be investigated.

Glucose Transport Assay—Insulin Independent—3T₃-L1 fibroblast are platedin 96-well plates and differentiated into fat cells (adipocytes) for 2weeks. After differentiation they are starved in serum-free medium andtreated with FGF-21 for 24 hours. Upon treatment, cells are washed twicewith KRBH buffer, containing 0.1% BSA. Glucose uptake is performed inthe presence of labeled glucose (without insulin) in KPBH buffer. FGF-21has been shown to stimulate glucose uptake in 3T₃-L1 adipocytes in aconcentration dependent manner at a sub-optimal concentration of insulin(5 nM) and in the absence of insulin (see US Patent Publication No.2004259780). Additionally, FGF-21 polypeptides of the present inventionmay be shown to induce glucose uptake in an ex vivo tissue model.

In the ex vivo glucose transport model, the glucose transport assay isdescribed as follows: Krebs-Henseleit Buffer Stock Solutions—Stock 1:NaCl (1.16 M); KCl (0.046 M); KH₂PO₄ (0.0116 M); NaHCO₃(0.0253 M). Stock2: CaCl₂ (0.025 M); MgSO₄ (2H₂O) (0.0116 M). BSA: Use ICN Cohn FractionV, fatty acid free BSA directly without dialysing. Media Preparation:Add 50 ml of Krebs stock 1 to 395 ml of dH₂O and gas with 95% O₂/5% CO₂for 1 hour. Add 50 ml of stock 2 and bring to 500 ml with dH₂O. Add 500mg of ICN fatty acid free BSA. Preincubation and Incubation Media: 32 mMMannitol, 8 mM Glucos. Wash Media: 40 mM Mannitol, 2 mM Pyruvate.Transport Media: 39 mM Mannitol, 1 mM 2-DG; 32 mM Mannitol, 8 mM 3-O-MG.Insulin Solution: (Porcine Insulin [Lilly] 100,000,000 μU/ml) at a finalconcentration of 2000 μU/ml or 13.3 nM. Radioactive Label MediaPreparation: Specific activities used: 2DG=1.5 mCi/ml; 3-O-MG=437μCi/ml; or, Mannitol=8 μCi/m. Rats are anesthetized with 0.1 cc Nembutalper 100 g body weight. Muscle tissue is excised and rinsed in 0.9%saline then placed in pre-incubation media (2 ml) at 29° C. for 1 hour.The muscle tissue is transferred to incubation media (2 ml; same aspre-incubation except including insulin or test compound) and incubatedfor 30 minutes (depends upon experimental conditions). The muscle tissueis then transferred to wash media (2 ml) for 10 minutes at 29° C., thentransferred to label media (1.5 ml) for 10 min (3-O-MG) or 20 min (2DG).The muscle tissue is trimmed, weighed and placed in polypropylene tubeson dry ice. 1 ml of 1 N KOH is added to the tubes which are then placedin a 70° C. water bath for 10-15 minutes, vortexing the tubes every fewminutes. The tubes are cooled on ice and 1 ml of 1 N HCl is added, thenmixed well. 200 μl of supernatant is then put in duplicate scintillationvials and counted on a scintillation counter compared to knownradioactive standards.

For contraction, the muscles are first incubated for 1 hour inpreincubation/incubation media. After 1 hour, one muscle of each pair(one pair per rat) is pinned to the stimulation apparatus and the othermuscle is transferred to a new flask of incubation media. The contractedmuscle is stimulated by 200 msec trains of 70 Hz with each impulse in atrain being 0.1 msec. The trains are delivered at 1/sec at 10-15V for2×10 minutes with a 1 minute rest in between. At the end of thestimulation period, the muscle is removed from the stimulation apparatusand placed in wash media for 10 minutes, followed by label media asoutlined above.

The FGF receptor can be prepared using techniques and methods that areknown to one of ordinary skill in the art. FGF-21 polypeptide activitycan be determined using standard or known in vitro or in vivo assays.For a non-PEGylated or PEGylated FGF-21 polypeptide comprising anon-natural amino acid, the affinity of FGF-21 for its receptor can bemeasured by using a BIAcore™ biosensor (Pharmacia).

Regardless of which methods are used to create the FGF-21 polypeptides,the FGF-21 polypeptides are subject to assays for biological activity.In general, the test for biological activity should provide analysis forthe desired result, such as increase or decrease in biological activity(as compared to modified FGF-21), different biological activity (ascompared to modified FGF-21), receptor or binding partner affinityanalysis, conformational or structural changes of the FGF-21 itself orits receptor (as compared to the modified FGF-21), or serum half-lifeanalysis.

The above compilation of references for assay methodologies is notexhaustive, and those of ordinary skill in the art will recognize otherassays useful for testing for the desired end result.

XIII. Measurement of Potency, Functional In Vivo Half-Life, andPharmacokinetic Parameters

An important aspect of the invention is the prolonged biologicalhalf-life that is obtained by construction of the FGF-21 polypeptidewith or without conjugation of the polypeptide to a water solublepolymer moiety. The rapid post administration decrease of FGF-21polypeptide serum concentrations has made it important to evaluatebiological responses to treatment with conjugated and non-conjugatedFGF-21 polypeptide and variants thereof. The conjugated andnon-conjugated FGF-21 polypeptide and variants thereof of the presentinvention may have prolonged serum half-lives also after administrationvia, e.g. subcutaneous or i.v. administration, making it possible tomeasure by, e.g. ELISA method or by a primary screening assay. ELISA orRIA kits from commercial sources may be used. Measurement of in vivobiological half-life is carried out as described herein.

The potency and functional in vivo half-life of an FGF-21 polypeptidecomprising a non-naturally encoded amino acid can be determinedaccording to protocols known to those of ordinary skill in the art.

Pharmacokinetic parameters for a FGF-21 polypeptide comprising anon-naturally encoded amino acid can be evaluated in normalSprague-Dawley male rats (N═5 animals per treatment group). Animals willreceive either a single dose of 25 ug/rat iv or 50 ug/rat sc, andapproximately 5-7 blood samples will be taken according to a pre-definedtime course, generally covering about 6 hours for a FGF-21 polypeptidecomprising a non-naturally encoded amino acid not conjugated to a watersoluble polymer and about 4 days for a FGF-21 polypeptide comprising anon-naturally encoded amino acid and conjugated to a water solublepolymer. Pharmacokinetic data for FGF-21 without a non-naturally encodedamino acid can be compared directly to the data obtained for FGF-21polypeptides comprising a non-naturally encoded amino acid.

Pharmacokinetic parameters can also be evaluated in a primate, e.g.,cynomolgus monkeys. Typically, a single injection is administered eithersubcutaneously or intravenously, and serum FGF-21 levels are monitoredover time.

The specific activity of FGF-21 polypeptides in accordance with thisinvention can be determined by various assays known in the art. Thebiological activity of the FGF-21 polypeptide muteins, or fragmentsthereof, obtained and purified in accordance with this invention can betested by methods described or referenced herein or known to those ofordinary skill in the art.

Polypeptides of the present invention may be used to treat mammalssuffering from non-insulin dependent Diabetes Mellitus (NIDDM: Type 2),insulin dependent diabetes (Type 1), as well as obesity, inadequateglucose clearance, hyperglycemia, hyperinsulinemia, and the like. FGF-21is effective in animal models of diabetes and obesity, as shown in USPatent Publication No. 20040259780, which is incorporated by referenceherein in its entirety. As metabolic profiles differ among variousanimal models of obesity and diabetes, analysis of multiple models havebeen undertaken to separate the effects of hyperinsulinemia,hyperglycemia and obesity. The diabetes (db/db) and obese (ob/ob) miceare characterized by massive obesity, hyperphagia, variablehyperglycemia, insulin resistance, hyperinsulinemia and impairedthermogenesis (Coleman, Diabetes 31:1, 1982; E. Shafrir, in DiabetesMellitus; H. Rifkin and D. Porte, Jr. Eds. (Elsevier Science PublishingCo., Inc., New York, ed. 4, 1990), pp. 299-340). However, diabetes ismuch more severe in the db/db model (Coleman, Diabetes 31:1, 1982; E.Shafrir, in Diabetes Mellitus; H. Rifkin and D. Porte, Jr. Eds.(Elsevier Science Publishing Co., Inc., New York, ed. 4, 1990), pp.299-340). Zucker (fa/fa) rats are severely obese, hyperinsulinemic, andinsulin resistant (Coleman, Diabetes 31:1, 1982; E. Shafrir, in DiabetesMellitus; H. Rifkin and D. Porte, Jr. Eds. (Elsevier Science PublishingCo., Inc., New York, ed. 4, 1990), pp. 299-340), and the fa/fa mutationmay be the rat equivalent of the murine db mutation (Friedman et al.,Cell 69:217-220, 1992; Truett et al., Proc. Natl. Acad. Sci. USA88:7806, 1991). Tubby (tub/tub) mice are characterized by obesity,moderate insulin resistance and hyperinsulinemia without significanthyperglycemia (Coleman et al., J. Heredity 81:424, 1990).

The monosodium glutamate (MSG) model for chemically-induced obesity(Olney, Science 164:719, 1969; Cameron et al., Cli. Exp. Pharmacol.Physiol. 5:41, 1978), in which obesity is less severe than in thegenetic models and develops without hyperphagia, hyperinsulinemia andinsulin resistance, may also be examined. Finally, the streptozotocin(STZ) model for chemically-induced diabetes may be tested to examine theeffects of hyperglycemia in the absence of obesity. STZ-treated animalsare deficient in insulin and severely hyperglycemic (Coleman, Diabetes31:1, 1982; E. Shafrir, in Diabetes Mellitus; H. Rifkin and D. Porte,Jr. Eds. (Elsevier Science Publishing Co., Inc., New York, ed. 4, 1990),pp. 299-340).

FGF-21 polypeptides of the invention can be evaluated in an in vivoseptic shock model in ob/ob mice. See U.S. Patent Publication No.20050176631, which is incorporated by reference in its entirety herein.

XIV. Administration and Pharmaceutical Compositions

The polypeptides or proteins of the invention (including but not limitedto, FGF-21, synthetases, proteins comprising one or more unnatural aminoacid, etc.) are optionally employed for therapeutic uses, including butnot limited to, in combination with a suitable pharmaceutical carrier.Such compositions, for example, comprise a therapeutically effectiveamount of the compound, and a pharmaceutically acceptable carrier orexcipient. Such a carrier or excipient includes, but is not limited to,saline, buffered saline, dextrose, water, glycerol, ethanol, and/orcombinations thereof. The formulation is made to suit the mode ofadministration. In general, methods of administering proteins are knownto those of ordinary skill in the art and can be applied toadministration of the polypeptides of the invention.

Therapeutic compositions comprising one or more polypeptide of theinvention are optionally tested in one or more appropriate in vitroand/or in vivo animal models of disease, to confirm efficacy, tissuemetabolism, and to estimate dosages, according to methods known to thoseof ordinary skill in the art. In particular, dosages can be initiallydetermined by activity, stability or other suitable measures ofunnatural herein to natural amino acid homologues (including but notlimited to, comparison of a FGF-21 polypeptide modified to include oneor more unnatural amino acids to a natural amino acid FGF-21polypeptide), i.e., in a relevant assay.

Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells. The unnaturalamino acid polypeptides of the invention are administered in anysuitable manner, optionally with one or more pharmaceutically acceptablecarriers. Suitable methods of administering such polypeptides in thecontext of the present invention to a patient are available, and,although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective action or reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention.

FGF-21 polypeptides of the invention may be administered by anyconventional route suitable for proteins or peptides, including, but notlimited to parenterally, e.g. injections including, but not limited to,subcutaneously or intravenously or any other form of injections orinfusions. Polypeptide compositions can be administered by a number ofroutes including, but not limited to oral, intravenous, intraperitoneal,intramuscular, transdermal, subcutaneous, topical, sublingual, or rectalmeans. Compositions comprising non-natural amino acid polypeptides,modified or unmodified, can also be administered via liposomes. Suchadministration routes and appropriate formulations are generally knownto those of skill in the art. The FGF-21 polypeptide, may be used aloneor in combination with other suitable components such as apharmaceutical carrier. The FGF-21 polypeptide may be used incombination with other agents, including but not limited to, an oralanti-diabetic agent.

The term “anti-diabetic agent” shall mean any drug that is useful intreating, preventing, or otherwise reducing the severity of any glucosemetabolism disorder, or any complications thereof, including any of theconditions, disease, or complications described herein. Anti-diabeticagents include insulin, thiazolidinediones, sulfonylureas, benzoic acidderivatives, alpha-glucosidase inhibitors, or the like. Other generalcategories of anti-diabetic agents which may be part of a subjectcomposition include (with defined terms being in quotation marks): “drugarticles” recognized in the official United States Pharmacopoeia orofficial National Formulary (or any supplement thereto); “new drug” and“new animal drug” approved by the FDA of the U.S. as those terms areused in Title 21 of the United States Code; any drug that requiresapproval of a government entity, in the U.S. or abroad (“approveddrug”); any drug that it is necessary to obtain regulatory approval soas to comply with 21 U.S.C. .sctn.355(a) (“regulatory approved drug”);any agent that is or was subject to a human drug application under 21U.S.C. .sctn.379(g) (“human drug”). (All references to statutory codefor this definition refer to such code as of the original filing date ofthis application.) Other anti-diabetic agents are disclosed herein, andare known to those of skill in the art. Current drugs or anti-diabeticagents used for managing Type 2 diabetes that are well-known in the artinclude a number of categories: the biguanides, thiazolidinediones, thesulfonylureas, benzoic acid derivatives and glucosidase inhibitors.These drugs usually have distinct modes of action. The biguanides, e.g.,metformin, are believed to prevent excessive hepatic gluconeogenesis.The thiazolidinediones are believed to act by increasing the rate ofperipheral glucose disposal. The sulfonylureas, e.g., tolbutamide andglyburide, and the benzoic acid derivatives, e.g. repaglinide, lowerplasma glucose by stimulating insulin secretion. The alpha-glucosidaseinhibitors competitively inhibit alpha-glucosidase, which metabolizescarbohydrates, thereby delaying carbohydrate absorption and attenuatingpost-prandial hyperglycemia. In addition, there are a number of proposedtherapies for treatment of diabetes that have not yet been approved forhuman use.

Current drugs or anti-diabetic agents used for managing diabetes and itsprecursor syndromes, such as insulin resistance, that are well-known inthe art include five classes of compounds: the biguanides, e.g.,metformin; thiazolidinediones; the sulfonylureas, e.g., tolbutamide andglyburide; benzoic acid derivatives, e.g. repaglinide; and glucosidaseinhibitors. In addition to these agents, a number of other therapies maybe used in combination with the FGF-21 polypeptides of the presentinvention to improve glucose control, including but not limited to DPP-4inhibitors. Certain of these anti-diabetic agents have been approved forhuman use. The lead DPP-4 compounds tested in clinical trials includeVildagliptin (Galvus) (LAF237), Sitagliptin (Januvia), Saxagliptin andAlogliptin. Januvia (Sitagliptin) was approved for the treatment of type2 diabetes in the United States on Oct. 17, 2006, for use asmonotherapy, or combination therapy, with either metformin or athiazolidinedione. Administration of the first generation Novartiscompound 1-[[[2-[(5-cyanopyridin-2-yl)amino]ethyl]amino]acetyl]-2-cyano-(S)-pyrrolidine (NVP DPP728) over a 4 weekperiod to 93 patients with Type 2 diabetes (mean HbA1c of 7.4%) reducedlevels of plasma glucose, insulin, and HbA1c over the 4 week studyperiod. See Inhibition of Dipeptidyl Peptidase IV Improves MetabolicControl Over a 4-Week Study Period in Type 2 Diabetes. Diabetes Care.2002 May; 25(5):869-875. Patients receiving metformin have also beennoted to exhibit additive glucose lowering benefits followinginstitution of GLP-1 therapy. See Additive glucose-lowering effects ofglucagon-like peptide-1 and metformin in type 2 diabetes. Diabetes Care.2001 Apr.; 24(4): 720-5. In a study of 10 obese non-diabetic malepatients, metformin administration was associated with increased levelsof circulating GLP-1 following oral glucose-loading, and in experimentsusing pooled human plasma, metformin (0.1-0.5 microg/ml) significantlyinhibited degradation of GLP-1(7-36)amide after a 30-min incubation at37 degrees C., in the presence or absence of DPP-4. The authors of thisstudy raised the possibility that metformin may inhibit the enzymaticbreakdown of GLP-1 both in vitro and in vivo. See Effect of metformin onglucagon-like peptide 1 (GLP-1) and leptin levels in obese nondiabeticsubjects. Diabetes Care. 2001 Mar.; 24(3):489-94. Analysis of therelationship between DPP-4, and GLP-1 degradation using biochemicalanalyses in vitro. Demuth and colleagues found no effect of metformin onthe DPP-4-mediated degradation of GLP-1 using a variety of sources ofhuman DPP-4. See Metformin Effects on Dipeptidylpeptidase IV Degradationof Glucagon-like Peptide-1. Biochem Biophys Res Commun. 2002 Mar. 15;291(5): 1302-8.

Among biguanides useful as diabetic therapeutic agents, metformin hasproven particularly successful. Metformin(N,N-dimethylimidodicarbonimidicdiamide; 1,1-dimethylbiguanide;N,N-dimethylbiguanide; N,N-dimethyldiguanide;N′-dimethylguanylguanidine) is an anti-diabetic agent that acts byreducing glucose production by the liver and by decreasing intestinalabsorption of glucose. It is also believed to improve the insulinsensitivity of tissues elsewhere in the body (increases peripheralglucose uptake and utilization). Metformin improves glucose tolerance inimpaired glucose tolerance (IGT) subjects and Type 2 diabetic subjects,lowering both pre- and post-prandial plasma glucose. Metformin isgenerally not effective in the absence of insulin. Bailey, Diabetes Care15:755-72 (1992).

The efficacy of metformin has been shown in several trials. In one studyof moderately obese Type 2 diabetics, HbA1c levels improved from 8.6% to7.1% after 29 weeks of metformin therapy alone or in combination withsulfonylurea. DeFronzo et al., New Engl. J. Med. 333:541-49 (1995).Metformin also had a favorable effect on serum lipids, lowering meanfasting serum triglycerides, total cholesterol, and LDL cholesterollevels and showing no adverse effects on other lipid levels. In anothertrial, metformin improved glycemic control in NIDDM subjects in adose-related manner. After 14 weeks, metformin 500 and 2000 mg dailyreduced HbA1c by 0.9% and 2.0%, respectively. Garber et al., Am J. Med.102:491-97 (1997). Metformin may also have a beneficial therapeuticeffect on insulin resistant non-diabetics. One study indicated thattreatment of hypertensive obese non-diabetic women with metformindecreased blood pressure, fasting and glucose-stimulated plasma insulinfibrinogen. Giugliano et al., Diabetes Care 16:1387-90 (1993).

Metformin is commonly administered as metformin HCl. This as well as allother useful forms of metformin are contemplated for use with FGF-21polypeptides of the present invention. Generally, a fixed dosage regimenis individualized for the management of hyperglycemia in diabetes withmetformin HCl or any other pharmacologic agent. Individualization ofdosage is made on the basis of both effectiveness and tolerance, whilegenerally not exceeding the maximum recommended daily dose of 2550 mg.

Thiazolidinediones contemplated for use in the practice of the presentinvention include troglitazone, and the like. Such compounds arewell-known, e.g., as described in U.S. Pat. Nos. 5,223,522, 5,132,317,5,120,754, 5,061,717, 4,897,405, 4,873,255, 4,687,777, 4,572,912,4,287,200, and 5,002,953; and Current Pharmaceutical Design 2:85-101(1996). Troglitazone is an oral antihyperglycemic agent that increasesglucose transport possibly by activation of peroxisomeproliferator-activated receptor-γ (PPARy). By such activation,troglitazone may enhance expression of GLUT4 glucose transporters,resulting in increased insulin-stimulated glucose uptake. Troglitazonemay also attenuate gluconeogenesis and/or activation of glycolysis.

HbA1c is a blood test that measures the amount of glycosylated which isgenerally higher when a patient has experienced periods of increasedblood glucose. The test provides an estimate of the last 2-3 months ofdiabetes management for a patient. Glycemic control resulting fromtroglitazone therapy reduces HbA1c by approximately 1 to 2%. Mimura etal., Diabetes Med. 11:685-91 (1994); Kumar et al., Diabetologia39:701-09 (1996). Effects may not occur for a few weeks after beginningtherapy. Troglitazone may also decrease insulin requirements. In onetrial of patients with NIDDM and using exogenous insulin, mean HbA1cfell by 0.8% and 1.4% for doses of 200 and 600 mg troglitazone,respectively. Insulin requirements were reduced by up to 29%. Schwartzet al., New Engl. J. Med. 338:861-66 (1998). In another study of NIDDMdiabetics using 400 and 600 mg troglitazone, fasting and post-prandialglucose levels were decreased, and hyperinsulinemic euglycemic clamindicated that glucose disposal was approximately 45% above pretreatmentlevels. Maggs et al., Ann. Intern. Med. 128:176-85 (1998).

In one study, 400 mg of troglitazone increased glucose disposal rates inobese patients with either impaired or normal glucose tolerance. Nolanet al., New Eng. J. Med 331:1188-93 (1994). In another study of womenwith IGT and a history of gestational diabetes, 600 mg troglitazoneimproved insulin homeostasis, including improving insulin sensitivityand lowering circulating insulin concentrations, but glucose tolerancewas unchanged. Berkowitz et al., Diabetes 45:172-79 (1996).Thiazolidinediones may be used with at-risk populations for NIDDM, suchas women with POCS or GDM, to prevent or delay the onset of NIDDM. U.S.Pat. No. 5,874,454. Effective amounts of troglitazone, when used alone,range from about 10 mg up to about 800 mg per daily dose and acommensurate range is contemplated for use in the present invention. Inaddition to being used with metformin, troglitazone may be used incombination with insulin and a sulfonylurea agent. See, for example,U.S. Pat. No. 5,859,037.

Sulfonylureas generally operate by lowering plasma glucose by increasingthe release of insulin from the pancreas. Specifically, sulfonylureasact by blocking the ATP-sensitive potassium channels. The sulfonylureaglimepiride may also increase insulin sensitivity by stimulatingtranslocation of GLUT4 transporters. Sulfonylureas are typicallyprescribed when HbA1c is above 8%. See also U.S. Pat. Nos. 5,258,185,4,873,080.

The sulfonylureas are a class of compounds that are well-known in theart, e.g., as described in U.S. Pat. Nos. 3,454,635, 3,669,966,2,968,158, 3,501,495, 3,708,486, 3,668,215, 3,654,357, and 3,097,242.Exemplary sulfonylureas contemplated for use in certain embodiments ofthe present invention (with typical daily dosages indicated inparentheses) include acetohexamide (in the range of about 250 up toabout 1500 mg), chlorpropamide (in the range of about 100 up to about500 mg), tolazimide (in the range of about 100 up to about 1000 mg),tolbutamide (in the range of about 500 up to about 3000 mg), gliclazide(in the range of about 80 up to about 320 mg), glipizide (in the rangeof about 5 up to about 40 mg), glipizide GITS (in the range of about 5up to about 20 mg), glyburide (in the range of about 1 up to about 20mg), micronized glyburide (in the range of about 0.75 up to about 12mg), glimeperide (in the range of about 1 up to about 8 mg), AG-EE 623ZW, and the like. Glimepiride is the first anti-diabetic agent in thisclass to be approved for use with insulin, and there may be less risk ofhypoglycemia associated with its use

A variety of alpha-glucosidase inhibitors may used with the presentinvention to treat and/or prevent diabetes. Such inhibitorscompetitively inhibit alpha-glucosidase, which metabolizescarbohydrates, thereby delaying carbohydrate absorption and attenuatingpost-prandial hyperglycemia. Clissod et al., Drugs 35:214-23 (1988). Thedecrease in glucose may be shown through decreased HbA1c levels.Exemplary alpha-glucosidase inhibitors contemplated for use in thepractice of the present invention include acarbose, miglitol, and thelike. Effective dosages of both acarbose and miglitol are in the rangeof about 25 up to about 300 mg daily.

Alpha-glucosidase inhibitors may be used with polypeptides of thepresent invention in combination with sulfonylureas. Alpha-glucosidaseinhibitors in combination with sulfonylureas alone have been shown todecrease HbA1c levels generally, from about 0.5 to 1.0%. In addition,alpha-glucosidase inhibitors have been shown to be effective in reducingthe post-prandial rise in blood glucose. Lefevre et al., Drugs 44:29-38(1992).

A variety of benzoic acid derivatives may used with polypeptides of thepresent invention to treat and/or prevent diabetes. These agents, alsoknown as meglitinides, are non-sulfonylurea hypoglycemic agents havinginsulin secretory capacity. For example, repaglinide appears to bind toATP-sensitive potassium channels on pancreatic beta cells and therebyincreases insulin secretion. Exemplary benzoic acid derivativescontemplated for use in the practice of the present invention includerepaglinide and the like. For repaglinide, the effective daily dosagemay be in the range of about 0.5 mg up to about 16 mg, and the agent maybe taken before each meal.

A number of agents are presently under investigation as potentialanti-diabetics in humans. Any of such agents may be used withpolypeptides of the present invention for treatment and/or prevention ofmetabolic disorders, and in particular of diabetes, if they becomeavailable for therapeutic use.

Another category of anti-diabetic agents that is inhibitors of carnitinepalmitoyl-transferase I (CPT-I), such as etomoxir, which in anadditional embodiment of the invention may be used with the modifiedFGF-21 polypeptides to modulate blood glucose. Etomoxir irreversiblyinhibits carnitine palmitoyl-transferase I, which is necessary for fattyacid oxidation. Such inhibition may reduce fasting hyperglycemia,because products of fatty acid oxidation stimulate hepaticgluconeogenesis. Etomoxir may improve insulin sensitivity in Type 2diabetics. Hubinger et al., Hormone Metab. Res. 24:115-18 (1992).

Other known anti-diabetic agents include insulin preparations (e.g.,animal insulin preparations extracted from pancreas of bovine and swine;human insulin preparations genetically synthesized using Escherichiacoli, yeast; zinc insulin; protamine zinc insulin; fragment orderivative of insulin (e.g., INS-1), oral insulin preparation), insulinsensitizers (e.g., pioglitazone or a salt thereof (preferablyhydrochloride), rosiglitazone or a salt thereof (preferably maleate),Netoglitazone, Rivoglitazone (CS-011), FK-614, the compound described inWO01/38325, Tesaglitazar (AZ-242), Ragaglitazar (N,N-622), Muraglitazar(BMS-298585), Edaglitazone (BM-13-1258), Metaglidasen (MBX-102),Naveglitazar (LY-519818), MX-6054, LY-510929, AMG-131(T-131), THR-0921),α-glucosidase inhibitors (e.g., voglibose, acarbose, miglitol,emiglitate etc.), biguanides (e.g., phenformin, metformin, buformin or asalt thereof (e.g., hydrochloride, fumarate, succinate)), insulinsecretagogues [sulfonylurea (e.g., tolbutamide, glibenclamide,gliclazide, chlorpropamide, tolazamide, acetohexamide, glyclopyramide,glimepiride, glipizide, glybuzole), repaglinide, nateglinide,mitiglinide or calcium salt hydrate thereof], dipeptidyl peptidase IVinhibitors (e.g., Vidagliptin (LAF237), P32/98, Sitagliptin (MK-431),P93/01, PT-100, Saxagliptin (BMS-477118), T-6666, TS-021), .beta.3agonists (e.g., AJ-9677), GPR40 agonists, glucagon-like polypeptides (I)(glp I), (glp2), or other diabetogenic peptide hormones, GLP-1 receptoragonists [e.g., GLP-1, GLP-1MR agent, N,N-2211, AC-2993 (exendin-4),BIM-51077, Aib(8,35)hGLP-1 (7,37)NH.sub.2, CJC-[131], amylin agonists(e.g., pramlintide), phosphotyrosine phosphatase inhibitors (e.g.,sodium vanadate), gluconeogenesis inhibitors (e.g., glycogenphosphorylase inhibitors, glucose-6-phosphatase inhibitors, glucagonantagonists), SGLUT (sodium-glucose cotransporter) inhibitors (e.g.,T-1095), 11.beta.-hydroxysteroid dehydrogenase inhibitors (e.g.,BVT-3498), adiponectin or agonists thereof, IKK inhibitors (e.g.,AS-2868), leptin resistance improving drugs, somatostatin receptoragonists (compounds described in WO01/25228, WO03/42204, WO98/44921,WO98/45285, WO99/2273.5 etc.), glucokinase activators (e.g.,R.sup.o-28-1675), GIP (Glucose-dependent insulinotropic peptide) and thelike can be mentioned.

The FGF-21 polypeptide comprising a non-natural amino acid, alone or incombination with other suitable components, can also be made intoaerosol formulations (i.e., they can be “nebulized”) to be administeredvia inhalation. Aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations of FGF-21 can be presented in unit-dose or multi-dosesealed containers, such as ampules and vials.

Parenteral administration and intravenous administration are preferredmethods of administration. In particular, the routes of administrationalready in use for natural amino acid homologue therapeutics (includingbut not limited to, those typically used for EPO, GH, G-CSF, GM-CSF,IFNs, interleukins, antibodies, FGFs, and/or any other pharmaceuticallydelivered protein), along with formulations in current use, providepreferred routes of administration and formulation for the polypeptidesof the invention.

The dose administered to a patient, in the context of the presentinvention, is sufficient to have a beneficial therapeutic response inthe patient over time, or other appropriate activity, depending on theapplication. The dose is determined by the efficacy of the particularvector, or formulation, and the activity, stability or serum half-lifeof the unnatural amino acid polypeptide employed and the condition ofthe patient, as well as the body weight or surface area of the patientto be treated. The size of the dose is also determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, formulation, or the like in aparticular patient.

In determining the effective amount of the vector or formulation to beadministered in the treatment or prophylaxis of disease (including butnot limited to, cancers, inherited diseases, diabetes, AIDS, or thelike), the physician evaluates circulating plasma levels, formulationtoxicities, progression of the disease, and/or where relevant, theproduction of anti-unnatural amino acid polypeptide antibodies.

The dose administered, for example, to a 70 kilogram patient, istypically in the range equivalent to dosages of currently-usedtherapeutic proteins, adjusted for the altered activity or serumhalf-life of the relevant composition. The vectors or pharmaceuticalformulations of this invention can supplement treatment conditions byany known conventional therapy, including antibody administration,vaccine administration, administration of cytotoxic agents, naturalamino acid polypeptides, nucleic acids, nucleotide analogues, biologicresponse modifiers, and the like.

For administration, formulations of the present invention areadministered at a rate determined by the LD-50 or ED-50 of the relevantformulation, and/or observation of any side-effects of the unnaturalamino acid polypeptides at various concentrations, including but notlimited to, as applied to the mass and overall health of the patient.Administration can be accomplished via single or divided doses.

If a patient undergoing infusion of a formulation develops fevers,chills, or muscle aches, he/she receives the appropriate dose ofaspirin, ibuprofen, acetaminophen or other pain/fever controlling drug.Patients who experience reactions to the infusion such as fever, muscleaches, and chills are premedicated 30 minutes prior to the futureinfusions with either aspirin, acetaminophen, or, including but notlimited to, diphenhydramine. Meperidine is used for more severe chillsand muscle aches that do not quickly respond to antipyretics andantihistamines. Cell infusion is slowed or discontinued depending uponthe severity of the reaction.

Human FGF-21 polypeptides of the invention can be administered directlyto a mammalian subject. Administration is by any of the routes normallyused for introducing FGF-21 polypeptide to a subject. The FGF-21polypeptide compositions according to embodiments of the presentinvention include those suitable for oral, rectal, topical, inhalation(including but not limited to, via an aerosol), buccal (including butnot limited to, sub-lingual), vaginal, parenteral (including but notlimited to, subcutaneous, intramuscular, intradermal, intraarticular,intrapleural, intraperitoneal, inracerebral, intraarterial, orintravenous), topical (i.e., both skin and mucosal surfaces, includingairway surfaces) and transdermal administration, although the mostsuitable route in any given case will depend on the nature and severityof the condition being treated. Administration can be either local orsystemic. The formulations of compounds can be presented in unit-dose ormulti-dose sealed containers, such as ampoules and vials. FGF-21polypeptides of the invention can be prepared in a mixture in a unitdosage injectable form (including but not limited to, solution,suspension, or emulsion) with a pharmaceutically acceptable carrier.FGF-21 polypeptides of the invention can also be administered bycontinuous infusion (using, including but not limited to, minipumps suchas osmotic pumps), single bolus or slow-release depot formulations.

Formulations suitable for administration include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. Solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.

Freeze-drying is a commonly employed technique for presenting proteinswhich serves to remove water from the protein preparation of interest.Freeze-drying, or lyophilization, is a process by which the material tobe dried is first frozen and then the ice or frozen solvent is removedby sublimation in a vacuum environment. An excipient may be included inpre-lyophilized formulations to enhance stability during thefreeze-drying process and/or to improve stability of the lyophilizedproduct upon storage. Pikal, M. Biopharm. 3(9)26-30 (1990) and Arakawaet al. Pharm. Res. 8(3):285-291 (1991).

The spray drying of pharmaceuticals is also known to those of ordinaryskill in the art. For example, see Broadhead, J. et al., “The SprayDrying of Pharmaceuticals,” in Drug Dev. Ind. Pharm, 18 (11 & 12),1169-1206 (1992). In addition to small molecule pharmaceuticals, avariety of biological materials have been spray dried and these include:enzymes, sera, plasma, micro-organisms and yeasts. Spray drying is auseful technique because it can convert a liquid pharmaceuticalpreparation into a fine, dustless or agglomerated powder in a one-stepprocess. The basic technique comprises the following four steps: a)atomization of the feed solution into a spray; b) spray-air contact; c)drying of the spray; and d) separation of the dried product from thedrying air. U.S. Pat. Nos. 6,235,710 and 6,001,800, which areincorporated by reference herein, describe the preparation ofrecombinant erythropoietin by spray drying.

The pharmaceutical compositions and formulations of the invention maycomprise a pharmaceutically acceptable carrier, excipient, orstabilizer. Pharmaceutically acceptable carriers are determined in partby the particular composition being administered, as well as by theparticular method used to administer the composition. Accordingly, thereis a wide variety of suitable formulations of pharmaceuticalcompositions (including optional pharmaceutically acceptable carriers,excipients, or stabilizers) of the present invention (see, e.g.,Remington's Pharmaceutical Sciences, 17th ed. 1985)).

Suitable carriers include but are not limited to, buffers containingsuccinate, phosphate, borate, HEPES, citrate, histidine, imidazole,acetate, bicarbonate, and other organic acids; antioxidants includingbut not limited to, ascorbic acid; low molecular weight polypeptidesincluding but not limited to those less than about 10 residues;proteins, including but not limited to, serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers including but not limited to,polyvinylpyrrolidone; amino acids including but not limited to, glycine,glutamine, asparagine, arginine, histidine or histidine derivatives,methionine, glutamate, or lysine; monosaccharides, disaccharides, andother carbohydrates, including but not limited to, trehalose, sucrose,glucose, mannose, or dextrins; chelating agents including but notlimited to, EDTA and edentate disodium; divalent metal ions includingbut not limited to, zinc, cobalt, or copper; sugar alcohols includingbut not limited to, mannitol or sorbitol; salt-forming counter ionsincluding but not limited to, sodium and sodium chloride; and/ornonionic surfactants including but not limited to Tween™ (including butnot limited to, Tween 80 (polysorbate 80) and Tween 20 (polysorbate 20),Pluronics™ and other pluronic acids, including but not limited to, andother pluronic acids, including but not limited to, pluronic acid F68(poloxamer 188), or PEG. Suitable surfactants include for example butare not limited to polyethers based upon poly(ethyleneoxide)-poly(propylene oxide)-poly(ethylene oxide), i.e., (PEO—PPO-PEO),or poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide),i.e., (PPO-PEO-PPO), or a combination thereof. PEO—PPO-PEO andPPO-PEO-PPO are commercially available under the trade names Pluronics™,R-Pluronics™, Tetronics™ and R-Tetronics™ (BASF Wyandotte Corp.,Wyandotte, Mich.) and are further described in U.S. Pat. No. 4,820,352incorporated herein in its entirety by reference. Otherethylene/polypropylene block polymers may be suitable surfactants. Asurfactant or a combination of surfactants may be used to stabilizePEGylated FGF-21 against one or more stresses including but not limitedto stress that results from agitation. Some of the above may be referredto as “bulking agents.” Some may also be referred to as “tonicitymodifiers.” Antimicrobial preservatives may also be applied for productstability and antimicrobial effectiveness; suitable preservativesinclude but are not limited to, benzyl alcohol, benzalkonium chloride,metacresol, methyl/propyl parabene, cresol, and phenol, or a combinationthereof.

FGF-21 polypeptides of the invention, including those linked to watersoluble polymers such as PEG can also be administered by or as part ofsustained-release systems. Sustained-release compositions include,including but not limited to, semi-permeable polymer matrices in theform of shaped articles, including but not limited to, films, ormicrocapsules. Sustained-release matrices include from biocompatiblematerials such as poly(2-hydroxyethyl methacrylate) (Langer et al., J.Biomed. Mater. Res., 15: 267-277 (1981); Langer, Chem. Tech., 12: 98-105(1982), ethylene vinyl acetate (Langer et al., supra) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988), polylactides (polylacticacid) (U.S. Pat. No. 3,773,919; EP 58,481), polyglycolide (polymer ofglycolic acid), polylactide co-glycolide (copolymers of lactic acid andglycolic acid) polyanhydrides, copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers, 22, 547-556 (1983),poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitinsulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides,nucleic acids, polyamino acids, amino acids such as phenylalanine,tyrosine, isoleucine, polynucleotides, polyvinyl propylene,polyvinylpyrrolidone and silicone. Sustained-release compositions alsoinclude a liposomally entrapped compound. Liposomes containing thecompound are prepared by methods known per se: DE 3,218,121; Eppstein etal., Proc. Natl. Acad. Sci. U.S.A., 82: 3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci. U.S.A., 77: 4030-4034 (1980); EP 52,322; EP36,676; U.S. Pat. No. 4,619,794; EP 143,949; U.S. Pat. No. 5,021,234;Japanese Pat. Appln. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545;and EP 102,324. All references and patents cited are incorporated byreference herein.

Liposomally entrapped FGF-21 polypeptides can be prepared by methodsdescribed in, e.g., DE 3,218,121; Eppstein et al., Proc. Natl. Acad.Sci. U.S.A., 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci.U.S.A., 77: 4030-4034 (1980); EP 52,322; EP 36,676; U.S. Pat. No.4,619,794; EP 143,949; U.S. Pat. No. 5,021,234; Japanese Pat. Appln.83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Composition and size of liposomes are well known or able to be readilydetermined empirically by one of ordinary skill in the art. Someexamples of liposomes as described in, e.g., Park J W, et al., Proc.Natl. Acad. Sci. USA 92:1327-1331 (1995); Lasic D and Papahadjopoulos D(eds): MEDICAL APPLICATIONS OF LIPOSOMES (1998); Drummond D C, et al.,Liposomal drug delivery systems for cancer therapy, in Teicher B (ed):CANCER DRUG DISCOVERY AND DEVELOPMENT (2002); Park J W, et al., Clin.Cancer Res. 8:1172-1181 (2002); Nielsen U B, et al., Biochim. Biophys.Acta 1591(1-3):109-118 (2002); Mamot C, et al., Cancer Res. 63:3154-3161 (2003). All references and patents cited are incorporated byreference herein.

The dose administered to a patient in the context of the presentinvention should be sufficient to cause a beneficial response in thesubject over time. Generally, the total pharmaceutically effectiveamount of the FGF-21 polypeptide of the present invention administeredparenterally per dose is in the range of about 0.01 μg/kg/day to about100 μg/kg, or about 0.05 mg/kg to about 1 mg/kg, of patient body weight,although this is subject to therapeutic discretion. The frequency ofdosing is also subject to therapeutic discretion, and may be morefrequent or less frequent than the commercially available FGF-21polypeptide products approved for use in humans. Generally, a PEGylatedFGF-21 polypeptide of the invention can be administered by any of theroutes of administration described above.

In some embodiments, modified FGF-21 polypeptides of the presentinvention modulate the effect of an anti-diabetic agent. In anotherembodiment of the present invention, modified FGF-21 polypeptides may becoadministered with an anti-diabetic agent. In another embodiment of thepresent invention, modified FGF-21 polypeptides may be administeredbefore treatment with an anti-diabetic agent. In another embodiment ofthe present invention, modified FGF-21 polypeptides may be administeredfollowing treatment with an anti-diabetic agent. In another embodimentof the present invention, modified FGF-21 polypeptides arecoadministered with metformin. In another embodiment of the presentinvention, therapeutic treatment with modified FGF-21 polypeptides ofthe invention and metformin increase the ability of metformin tomodulate plasma glucose, in the presence or absence of insulin. Incombination therapy, metformin has been used with sulfonylureas,alpha-glucosidase inhibitors, troglitazeon, and insulin. Metformincombined with a sulfonylurea increases insulin sensitivity and may lowerplasma glucose. Alternatively, metformin with repaglinide may be moreeffective than glipizide, and at least as effective as glyburide, inmaintaining glycemic control over many months. Metformin withtroglitazone improves glucose control in excess of either agent alone.Inzucchi et al., New. Eng. J. Med. 338:867-72 (1998). In someembodiments, the FGF-21 polypeptides of the present invention arecoadministered with Klotho beta. In some embodiments, the FGF-21polypeptides of the present invention are coadministered with Klothobeta that includes one or more non-naturally encoded amino acids. Insome embodiments, the FGF-21 polypeptides of the present invention arecoadministered with Klotho beta and an anti-diabetic agent. In someembodiments, the FGF-21 polypeptides of the present invention arecoadministered with an anti-diabetic agent. In some embodiments, FGF-21polypeptides of the present invention are used in combination with oneor more of the following: Taurine, Alpha Lipoic Acid, an extract ofMulberry, Chromium, Glutamine, Enicostemma littorale Blume, Scopariadulcis, an extract of Tarragon and Andrographis paniculata. In someembodiments, FGF-21 polypeptides of the present invention are used incombination with one or more of the following: insulin preparations(e.g., animal insulin preparations extracted from pancreas of bovine andswine; human insulin preparations genetically synthesized usingEscherichia coli, yeast; zinc insulin; protamine zinc insulin; fragmentor derivative of insulin (e.g., INS-1), oral insulin preparation),insulin sensitizers (e.g., pioglitazone or a salt thereof (preferablyhydrochloride), rosiglitazone or a salt thereof (preferably maleate),Netoglitazone, Rivoglitazone (CS-011), FK-614, the compound described inWO01/38325, Tesaglitazar (AZ-242), Ragaglitazar (N,N-622), Muraglitazar(BMS-298585), Edaglitazone (BM-13-1258), Metaglidasen (MBX-102),Naveglitazar (LY-519818), MX-6054, LY-510929, AMG-131(T-131), THR-0921),α-glucosidase inhibitors (e.g., voglibose, acarbose, miglitol,emiglitate etc.), biguanides (e.g., phenformin, metformin, buformin or asalt thereof (e.g., hydrochloride, fumarate, succinate)), insulinsecretagogues [sulfonylurea (e.g., tolbutamide, glibenclamide,gliclazide, chlorpropamide, tolazamide, acetohexamide, glyclopyramide,glimepiride, glipizide, glybuzole), repaglinide, nateglinide,mitiglinide or calcium salt hydrate thereof], dipeptidyl peptidase IVinhibitors (e.g., Vidagliptin (LAF237), P32/98, Sitagliptin (MK-431),P93/01, PT-100, Saxagliptin (BMS-477118), T-6666, TS-021), .beta.3agonists (e.g., AJ-9677), GPR40 agonists, glucagon-like polypeptides (I)(glp I), (glp2), or other diabetogenic peptide hormones, GLP-1 receptoragonists [e.g., GLP-1, GLP-1MR agent, N,N-2211, AC-2993 (exendin-4),BIM-51077, Aib(8,35)hGLP-1 (7,37)NH. sub 0.2, CJC-[131], amylin agonists(e.g., pramlintide), phosphotyrosine phosphatase inhibitors (e.g.,sodium vanadate), gluconeogenesis inhibitors (e.g., glycogenphosphorylase inhibitors, glucose-6-phosphatase inhibitors, glucagonantagonists), SGLUT (sodium-glucose cotransporter) inhibitors (e.g.,T-1095), 11.beta.-hydroxysteroid dehydrogenase inhibitors (e.g.,BVT-3498), adiponectin or agonists thereof, IKK inhibitors (e.g.,AS-2868), leptin resistance improving drugs, somatostatin receptoragonists (compounds described in WO01/25228, WO03/42204, WO98/44921,WO98/45285, WO99/2273.5 etc.), glucokinase activators (e.g., R.sup.o-28-1675), GIP (Glucose-dependent insulinotropic peptide).

In some embodiments, polypeptides of the present invention are used incombination with insulin potentiators such as, including but not limitedto, Taurine, Alpha Lipoic Acid, an extract of Mulberry, Chromium,Glutamine, Enicostemma littorale Blume, Scoparia dulcis, an extract ofTarragon and Andrographis paniculata. In another embodiment, the presentinvention may comprise one or more of Isomalt, Trehalose or D-Mannose tofurther potentiate the secretion or activity of insulin. In anadditional embodiment, the insulin potentiator and polypeptides of thepresent invention are used in addition to another anti-diabetic agent.

One way in which the therapeutic efficacy of the polypeptides andcombined therapies including the present invention's polypeptides may bedetermined is through a reduction in patient HbA1c levels. In oneembodiment, polypeptides of the present invention lower HbA1c levels byat least a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or at least50% change from HbA1c levels two months prior to beginning therapy withmodified FGF-21 polypeptides, from three months prior to beginningtherapy with modified FGF-21 polypeptides, or by percentage changes froma baseline. In another embodiment, polypeptides of the present inventionadministered to a patient also being treated with an anti-diabetic agentmodulate the ability of the anti-diabetic agent to lower blood glucose.In another embodiment, polypeptides of the present inventionadministered to a patient also being treated with an anti-diabetic agentlower HbA1c levels by at least a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%,40%, 45%, 50%, or at least 50% change from HbA1c levels two months priorto beginning therapy with modified FGF-21 polypeptides, from threemonths prior to beginning therapy with modified FGF-21 polypeptides, orby percentage changes from a baseline or from a pre-treatment baseline.

In another embodiment, modified FGF-21 polypeptides of the presentinvention modulate the ability of Troglitazone to decrease insulinrequirements. In another embodiment, Additional embodiment, modifiedFGF-21 polypeptides of the present invention when administered to apatient being treated with Troglitazone further decrease said patient'sinsulin requirements. Troglitazone used in combination with the presentinvention may be used to delay or prevent Type 2 diabetes in certainembodiments of the present invention.

In one embodiment of the present invention, modified FGF-21 polypeptidesare coadministered with a sulfonylurea. In another embodiment of thepresent invention, modified FGF-21 polypeptides are administered beforetreatment with a sulfonylurea. In another embodiment of the presentinvention, modified FGF-21 polypeptides are administered after treatmentwith a sulfonylurea. In some embodiments of the present invention,treatment with a therapeutic dose of modified FGF-21 polypeptidesmodulates serum glucose. In another embodiment, FGF-21 polypeptides ofthe present invention are administered with Klotho beta which modulatesthe effects of the polypeptides on blood glucose. In another embodiment,FGF-21 polypeptides of the present invention are administered withKlotho beta which decreases blood glucose more than use of modifiedFGF-21 polypeptides alone. In another embodiment, these changes aremeasured using HbA1c tests. In another embodiment, FGF-21 polypeptidesof the present invention and Klotho beta are administered to a patientbeing treated with an anti-diabetic agent which decreases blood glucosemore than use of the anti-diabetic agent alone.

XV. Therapeutic Uses of FGF-21 Polypeptides of the Invention

The FGF-21 polypeptides of the invention are useful for treating a widerange of disorders.

FGF-21 polypeptides of the invention may be used to treat mammalssuffering from non-insulin dependent Diabetes Mellitus (NIDDM: Type 2),insulin dependent diabetes (Type 1), as well as obesity, inadequateglucose clearance, hyperglycemia, hyperinsulinemia, and any otherdisease or condition that may be mediated by FGF-21. Glucose intolerancecan be defined as an exceptional sensitivity to glucose. Hyperglycemiais defined as an excess of sugar (glucose) in the blood.Hyperinsulinemia is defined as a higher-than-normal level of insulin inthe blood. Insulin resistance is defined as a state in which a normalamount of insulin produces a subnormal biologic response. Obesity, interms of the human subject, can be defined as that body weight over 20percent above the ideal body weight for a given population (R. H.Williams, Textbook of Endocrinology, 1974, p. 904-916).

Diabetes mellitus is characterized in two broad groups based on clinicalmanifestations, namely, the non-insulin-dependent or maturity onsetform, also known as Type 2; and the insulin-dependent or juvenile onsetform, also known as Type 1. Clinically, the majority of Type 2, maturityonset diabetics are obese, with manifestations of clinical symptoms ofthe disease usually appearing at an age over 40. In contrast, Type 1,juvenile onset patients are not over-weight relative to their age andheight, with rapid onset of the disease at an early age, often before30, although Type 1 diabetes can occur at any age.

Diabetes mellitus is a metabolic disorder in humans with a prevalence ofapproximately one percent in the general population, with one-fourth ofthese being the Type 1 (Foster, D. W., Harrison's Principles of InternalMedicine, Chap. 114, pp. 661-678, 10th Ed., McGraw-Hill, New York). Thedisease manifests itself as a series of hormone-induced metabolicabnormalities that eventually lead to serious, long-term anddebilitating complications involving several organ systems including theeyes, kidneys, nerves, and blood vessels. Pathologically, the disease ischaracterized by lesions of the basement membranes, demonstrable underelectron microscopy.

Non-insulin-dependent Diabetes Mellitus (NIDDM: Type 2) is adebilitating disease characterized by high-circulating blood glucose,insulin and corticosteroid levels. The incidence of Type 2 diabetes ishigh and rising and is becoming a leading cause of mortality, morbidityand healthcare expenditure throughout the world (Amos et al., DiabeticMed. 14:S1-85, 1997).

The causes of Type 2 diabetes are not well understood. Type 2 diabetesis characterized by excess glucose production in spite of theavailability of insulin, and circulating glucose levels remainexcessively high as a result of inadequate glucose clearance. It isthought that both resistance of target tissues to the action of insulinand decreased insulin secretion (“I3-cell failure”) occur. Majorinsulin-responsive tissues for glucose homeostasis are liver, in whichinsulin stimulates glycogen synthesis and inhibits gluconeogenesis;muscle, in which insulin stimulates glucose uptake and glycogenstimulates glucose uptake and inhibits lipolysis. Thus, as a consequenceof the diabetic condition, there are elevated levels of glucose in theblood, and prolonged high blood sugar which is indicative of a conditionwhich will cause blood vessel and nerve damage.

Currently, there are various pharmacological approaches for thetreatment of Type 2 diabetes (Scheen et al., Diabetes Care,22(9):1568-1577, 1999). They act via different modes of action: 1)sulfonyulureas essentially stimulate insulin secretion; 2) biguanides(metformin) act by promoting glucose utilization, reducing hepaticglucose production and diminishing intestinal glucose output; 3)a-glucosidase inhibitors (acarbose, miglitol) slow down carbohydratedigestion and consequently absorption from the gut and reducepostprandial hyperglycemia; 4) thiazol-idinediones (troglitazone)enhance insulin action, thus promoting glucose utilization in peripheraltissues; and 5) insulin stimulates tissue glucose utilization andinhibits hepatic glucose output. The above mentioned pharmacologicalapproaches may be utilized individually or in combination therapy.However, each approach has its limitations and adverse effects.

Obesity is a chronic disease that is highly prevalent in modern societyand is associated not only with a social stigma, but also with decreasedlife span and numerous medical problems including adverse psychologicaldevelopment, dermatological disorders such as infections, varicoseveins, exercise intolerance, diabetes mellitus, insulin resistance,hypertension, hypercholesterolemia, and coronary heart disease. Rissanenet al., British Medical Journal, 301: 835-837 (1990).

Existing therapies for obesity include standard diets and exercise, verylow calorie diets, behavioral therapy, pharmacotherapy involvingappetite suppressants, thermogenic drugs, food absorption inhibitors,mechanical devices such as jaw wiring, waist cords and balloons, andsurgery. Jung and Chong, Clinical Endocrinology, 35: 11-20 (1991); Bray,Am. J. Clin. Nutr., 55: 538S-544S (1992).

Considering the high prevalence of obesity in our society and theserious consequences associated therewith as discussed above, anytherapeutic drug potentially useful in reducing weight of obese personscould have a profound beneficial effect on their health. There is a needin the art for a drug that will reduce total body weight of obesesubjects toward their ideal body weight without significant adverse sideeffects and that will help the obese subject maintain the reduced weightlevel.

It is therefore desirable to provide a treatment regimen that is usefulin returning the body weight of obese subjects toward a normal, idealbody weight. It is further desirable to provide a therapy for obesitythat results in maintenance of the lowered body weight for an extendedperiod of time.

Obesity is highly correlated with insulin resistance and diabetes inexperimental animals and humans. Indeed, obesity and insulin resistance,the latter of which is generally accompanied by hyperinsulinemia orhyperglycemia, or both, are hallmarks of Type 2 diabetes. In addition,Type 2 diabetes is associated with a two- to fourfold risk of coronaryartery disease. Despite decades of research on these serious healthproblems, the etiology of obesity and insulin resistance is unknown.

Type 1 diabetics characteristically show very low or immeasurable plasmainsulin with elevated glucagon. Regardless of what the exact etiologyis, most Type 1 patients have circulating antibodies directed againsttheir own pancreatic cells including antibodies to insulin, to islet ofLangerhans cell cytoplasm and to the enzyme glutamic acid decarboxylase.An immune response specifically directed against beta cells (insulinproducing cells) leads to Type 1 diabetes. This specificity is supportedby the above clinical picture, since beta cells secrete insulin whilealpha cells secrete glucagon.

Current therapeutic regimens for Type 1 diabetes include modificationsto the diet in order to minimize hyper-glycemia resulting from the lackof natural insulin, which in turn, is the result of damaged beta cells.Diet is also modified with regard to insulin administration to counterthe hypoglycemic effects of the hormone. Whatever the form of treatment,parenteral administration of insulin is required for all Type 1diabetics, hence the term “insulin-dependent” diabetes.

Thus, there is a need for an effective therapy of Type 2 diabetes thathas fewer adverse effects than the available pharmaceutical approaches.Moreover, an effective alternative therapy to insulin could be usefulfor the treatment of Type 1 diabetes. The present invention provides apharmacological therapy which stimulates glucose uptake and enhancesinsulin sensitivity in peripheral tissues and has fewer adverse effectsthan current treatment regimens for Type 2 diabetes. In addition, thepresent invention provides an alternative treatment for Type 1 diabetes.Furthermore, the present invention is useful for treating obesity byincreasing energy expenditure by faster and more efficient glucoseutilization.

The present invention provides a method for treating a mammal exhibitingone or more of Type 1 diabetes, Type 2 diabetes, obesity, insulinresistance, hyperinsulinemia, glucose intolerance, or hyperglycemia,comprising administering to said mammal in need of such treatment atherapeutically effective amount of the FGF-21 polypeptide of theinvention.

The method of treating may be sufficient to achieve in said mammal atleast one of the following modifications: reduction in body fat stores,decrease in insulin resistance, reduction of hyperinsulinemia, increasein glucose tolerance, and reduction of hyperglycemia.

In another aspect, the present invention relates to a method of treatinga domestic animal including but not limited to, cattle, pigs, sheep,horses, and the like, comprising administering an effective amount ofFGF-21 or variant thereof, in order to reduce body fat stores. Thereduction of body fat stores on a long term, or permanent basis indomestic animals would obviously be of considerable economic benefit toman, particularly since animals supply a major portion of man's diet;and the animal fat may end up as de novo fat deposits in man.

Fibroblast growth factor 21 (FGF-21) may be used to reduce the morbidityand mortality associated with critically ill patients. See U.S. PatentPublication No. 20050176631 which is incorporated by reference herein inits entirety. Critically ill patients requiring intensive care for anextended period of time have a high risk of death and substantialmortality. A common cause for admittance of patients to intensive careunits (ICUs) is systemic inflammatory response syndrome (SIRS)associated with infectious insults (sepsis) as well as noninfectiouspathologic causes such as pancreatitis, ischemia, multiple trauma andtissue injury, hemorrhagic shock, and immune-mediated organ injury. Thepresent invention also encompasses a method of reducing mortality andmorbidity in critically ill patients suffering from systemicinflammatory response syndrome (SIRS) associated with infectious insultsas well as noninfectious pathologic causes which comprises administeringto the critically ill patients a therapeutically effective amount ofFGF-21. Examples of conditions that involve SIRS include sepsis,pancreatitis, ischemia, multiple trauma and tissue injury, hemorrhagicshock, immune-mediated organ injury, acute respiratory distress syndrome(ARDS), shock, renal failure, and multiple organ dysfunction syndrome(MODS). The present invention also encompasses a method of reducingmortality and morbidity in critically ill patients suffering fromrespiratory distress.

A frequent complication of SIRS is the development of organ systemdysfunction, including acute respiratory distress syndrome (ARDS),shock, renal failure, and multiple organ dysfunction syndrome (MODS),all of which amplify the risk of an adverse outcome. While manyspecialists believe that some type of nutritional support is beneficialto critically ill patients to help restore metabolic stability, thebenefits and specifics of such support remain controversial due to thelack of well-controlled randomized clinical trials.

Because hyperglycemia and insulin resistance are common in criticallyill patients given nutritional support, some ICUs administer insulin totreat excessive hyperglycemia in fed critically ill patients. In fact,recent studies document the use of exogenous insulin to maintain bloodglucose at a level no higher than 110 mg per deciliter reduced morbidityand mortality among critically ill patients in the surgical intensivecare unit, regardless of whether they had a history of diabetes (Van denBerghe, et al. N Engl J Med., 345(19):1359, 2001).

The present invention encompasses a method of reducing the mortality andmorbidity in these critically ill patients through the administration ofFGF-21. The critically ill patients encompassed by the present inventiongenerally experience an unstable hypermetabolic state. This unstablemetabolic state is due to changes in substrate metabolism which may leadto relative deficiencies in some nutrients. Generally there is increasedoxidation of both fat and muscle.

The critically ill patients wherein the administration of FGF-21 canreduce the risk of mortality and morbidity are preferably patients thatexperience systemic inflammatory response syndrome or respiratorydistress. A reduction in morbidity means reducing the likelihood that acritically ill patient will develop additional illnesses, conditions, orsymptoms or reducing the severity of additional illnesses, conditions,or symptoms. For example reducing morbidity may correspond to a decreasein the incidence of bacteremia or sepsis or complications associatedwith multiple organ failure.

Systemic inflammatory response syndrome (SIRS) describes an inflammatoryprocess associated with a large number of clinical conditions andincludes, but is not limited to, more than one of the following clinicalmanifestations: (1) a body temperature greater than 38° C. or less than36° C.; (2) a heart rate greater than 90 beats per minute; (3)tachypnea, manifested by a respiratory rate greater than 20 breaths perminute, or hyperventilation, as indicated by a PaCo2 of less than 32 mmHg; and (4) an alteration in the white blood cell count, such as a countgreater than 12,000/cu mm, a count less than 4,000/cu mm, or thepresence of more than 10% immature neutrophils. These physiologicchanges should represent an acute alteration from baseline in theabsence of other known causes for such abnormalities, such aschemotherapy, induced neutropenia, and leukopenia.

Sepsis is defined as a SIRS arising from infection. Noninfectiouspathogenic causes of SIRS may include pancreatitis, ischemia, multipletrauma and tissue injury, including but not limited to, crushinginjuries or severe burns, hemorrhagic shock, immune-mediated organinjury, and the exogenous administration of such putative mediators ofthe inflammatory process as tumor necrosis factor and other cytokines.

Septic shock and multi-organ dysfunction are major contributors tomorbidity and mortality in the ICU setting. Sepsis is associated withand mediated by the activation of a number of host defense mechanismsincluding the cytokine network, leukocytes, and the complement cascade,and coagulation/fibrinolysis systems including the endothelium.Disseminated intravascular coagulation (DIC) and other degrees ofconsumption coagulopathy associated with fibrin deposition within themicrovasculature of various organs, are manifestations of sepsis/septicshock. The downstream effects of the host defense response on targetorgans is an important mediator in the development of the multiple organdysfunction syndrome (MODS) and contributes to the poor prognosis ofpatients with sepsis, severe sepsis and sepsis complicated by shock.

Respiratory distress denotes a condition wherein patients havedifficulty breathing due to some type of pulmonary dysfunction. Oftenthese patients exhibit varying degrees of hypoxemia that may or may notbe refractory to treatment with supplemental oxygen. Respiratorydistress may occur in patients with impaired pulmonary function due todirect lung injury or may occur due to indirect lung injury such as inthe setting of a systemic process. In addition, the presence of multiplepredisposing disorders substantially increases the risk, as does thepresence of secondary factors such as chronic alcohol abuse, chroniclung disease, and a low serum pH.

Some causes of direct lung injury include pneumonia, aspiration ofgastric contents, pulmonary contusion, fat emboli, near-drowning,inhalation injury, high altitude and reperfusion pulmonary edema afterlung transplantation or pulmonary embolectomy. Some causes of indirectlung injury include sepsis, severe trauma with shock and multipletransfusions; cardiopulmonary bypass, drug overdose, acute pancreatitis,and transfusions of blood products.

One class of pulmonary disorders that causes respiratory distress areassociated with the syndrome known as Cor Pulmonale. These disorders areassociated with chronic hypoxemia resulting in raised pressure withinthe pulmonary circulation called pulmonary hypertension. The ensuingpulmonary hypertension increases the work load of the right ventricle,thus leading to its enlargement or hypertrophy. Cor Pulmonale generallypresents as right heart failure defined by a sustained increase in rightventricular pressures and clinical evidence of reduced venous return tothe right heart.

Chronic obstructive pulmonary diseases (COPDs) which include emphysemaand chronic bronchitis also cause respiratory distress and arecharacterized by obstruction to air flow. COPDs are the fourth leadingcause of death and claim over 100,000 lives annually.

Acute respiratory distress syndrome (ARDS) is generally progressive andcharacterized by distinct stages. The syndrome is generally manifestedby the rapid onset of respiratory failure in a patient with a riskfactor for the condition. Arterial hypoxemia that is refractory totreatment with supplemental oxygen is a characteristic feature. Theremay be alveolar filling, consolidation, and atelectasis occurring independent lung zones; however, non-dependent areas may have substantialinflammation. The syndrome may progress to fibrosing alveolitis withpersistent hypoxemia, increased alveolar dead space, and a furtherdecrease in pulmonary compliance. Pulmonary hypertension which resultsfrom damage to the pulmonary capillary bed may also develop.

The severity of clinical lung injury varies. Both patients with lesssevere hypoxemia as defined by a ratio of the partial pressure ofarterial oxygen to the fraction of inspired oxygen as 300 or less andpatients with more severe hypoxemia as defined by a ratio of 200 or lessare encompassed by the present invention. Generally, patients with aratio 300 or less are classified as having acute lung injury andpatients with having a ratio of 200 or less are classified as havingacute respiratory distress syndrome.

The acute phase of acute lung injury is characterized by an influx ofprotein-rich edema fluid into the air spaces as a consequence ofincreased vascular permeability of the alveolar-capillary barrier. Theloss of epithelial integrity wherein permeability is altered can causealveolar flooding, disrupt normal fluid transport which affects theremoval of edema fluid from the alveolar space, reduce the productionand turnover of surfactant, lead to septic shock in patients withbacterial pneumonia, and cause fibrosis. Sepsis is associated with thehighest risk of progression to acute lung injury.

In conditions such as sepsis, where hypermetabolism occurs, there is anaccelerated protein breakdown both to sustain gluconeogenesis and toliberate the amino acids required for increased protein synthesis.Hyperglycemia may be present and high concentrations of triglyceridesand other lipids in serum may be present.

For patients with compromised respiratory function, hypermetabolism mayaffect the ratio of carbon dioxide production to oxygen consumption.This is known as the respiratory quotient (R/Q) and in normalindividuals is between about 0.85 and about 0.90. Excess fat metabolismhas a tendency to lower the R/Q whereas excess glucose metabolism raisesthe R/Q. Patients with respiratory distress often have difficultyeliminating carbon dioxide and thus have abnormally high respiratoryquotients.

The critically ill patients encompassed by the present invention alsogenerally experience a particular stress response characterized by atransient down-regulation of most cellular products and theup-regulation of heat shock proteins. Furthermore, this stress responseinvolves the activation of hormones such as glucagon, growth hormone,cortisol, and pro- and anti-inflammatory cytokines. While this stressresponse appears to have a protective function, the response createsadditional metabolic instability in these critically ill patients. Forexample, activation of these specific hormones causes elevations inserum glucose which results in hyperglycemia. In addition, damage to theheart and other organs may be exacerbated by adrenergic stimuli.Further, there may be changes to the thyroid which may have significanteffects on metabolic activity.

Average quantities of the FGF-21 may vary and in particular should bebased upon the recommendations and prescription of a qualifiedphysician. The exact amount of FGF-21 is a matter of preference subjectto such factors as the exact type of condition being treated, thecondition of the patient being treated, as well as the other ingredientsin the composition. The invention also provides for administration of atherapeutically effective amount of another active agent. The amount tobe given may be readily determined by one of ordinary skill in the artbased upon therapy with FGF-21.

Pharmaceutical compositions of the invention may be manufactured in aconventional manner.

EXAMPLES

The following examples are offered to illustrate, but do not limit theclaimed invention.

Example 1

This example describes one of the many potential sets of criteria forthe selection of sites of incorporation of non-naturally encoded aminoacids into FGF-21.

FIG. 1 shows the sequence homology between FGF-21 (Protein accessionnumber BC018404) and FGF-19 (Protein accession number BAA75500) asdetermined using Vector NTI (Invitrogen; Carlsbad, Calif.). The aminoacids marked with an asterisk are similar between the two molecules. Theamino acids that are underlined are identical between the twopolypeptides. Seven different FGF-21 polypeptides were generated bysubstituting a naturally encoded amino acid with a non-naturally encodedamino acid. Each polypeptide had one of the amino acids marked with arectangle in FIG. 1 substituted with para-acetylphenylalanine. Thepolypeptides generated lacked the leader sequence shown in FIGS. 1 and 3and were His tagged at the N terminus with 6 histidine residues. SEQ IDNO.: 1 is a 181 amino acid sequence of human FGF-21 (P form) without theleader sequence. SEQ ID NO.: 2 is the sequence of human FGF-21 (P form)without the leader sequence and with a His tag at the N terminus. Eachof the polypeptides generated had a non-naturally encoded amino acidsubstitution at one of the following positions 10, 52, 77, 117, 126,131, and 162 of SEQ ID NO: 1.

FIG. 2 shows the structure of human FGF-19 that was obtained from theProtein Data Bank (PDB) (Bernstein et al. J. Mol. Biol. 1997, 112, pp535) (1PWA) and was labeled using the PyMOL software (DeLano Scientific;Palo Alto, Calif.). The amino acids corresponding to V34, L79, G104,5144, K155, L160, and 5196 of FGF-19 were substituted withpara-acetylphenylalanine in FGF-21 polypeptides of the invention. Thedashed line indicates regions that were not resolved in the originalstructure.

FIG. 3 shows the sequence homology between FGF-21 (Protein accessionnumber BC018404) and FGF-2 (Protein accession number BAA75500) asdetermined using Vector NTI (Invitrogen; Carlsbad, Calif.). The aminoacids marked with an asterisk are similar between the two molecules. Theamino acids that are underlined are identical between the twopolypeptides. The 7 amino acids shown in FIG. 1 as sites forsubstitution are also placed in a rectangle in FIG. 3.

FIG. 4 shows the structure of human FGF-2 structure that was obtainedfrom PDB (1CVS) and was labeled using the PyMOL software (DeLanoScientific; Palo Alto, Calif.). The gray structures are human FGFreceptor 1 (FGFR1) and the black is human FGF2. Plotnikov, A N et al.Cell. 1999 Sep. 3; 98(5):641-50 describe the crystal structure of FGF2bound to FGF receptor. The amino acids corresponding to F21, K62, K86,V125, K134, T₁₃₉ of FGF-2 were substituted with para-acetylphenylalaninein FGF-21 polypeptides of the invention. The dashed line indicatesregions that were not resolved in the original structure.

Another set of criteria for the selection of preferred sites ofincorporation of non-naturally encoded amino acids is the following. Tencrystal structures from the Protein Data Bank were used to model thestructure of FGF-21: 1PWA (human FGF-19); lIJT (human FGF-4); 1NUN(human FGF10-FGF Receptor 2b Complex); 1G82 (human FGF-9 dimer with FGFReceptor and heparin); 1IHK (human FGF-9); 1BAR (bovine FGF-1); 1QQK(rat FGF-7); 1K5U (human FGF-1); 1FQ9 (human FGF-2 with FGF Receptor 1and heparin); and 2FDB (human FGF-8b with FGF Receptor 2c). Thecoordinates for these structures are available from the Protein DataBank (PDB) (Bernstein et al. J. Mol. Biol. 1997, 112, pp 535). Acomparison of the crystal structures indicated that they were all verysimilar in the core structure. However, the N- and C-termini were foundto be highly divergent between these FGF molecules, and therefore thetermini could not be modeled. The modeling identified two residues, Y22and Y104, which were highly conserved and were involved with receptorbinding. Two potential heparin binding sites were also identifiedinvolving R₃₆ and E37. The amino acid positions identified for thereceptor binding and heparin binding residues correspond to SEQ ID NO:1.

As a result, residues were identified that 1) would not interfere withbinding to the FGF receptor or heparin, 2) would not be present in theinterior of the protein, and 3) would be in regions that were fairlyconsistent between the crystal structures. In some embodiments, one ormore non-naturally encoded encoded amino acids are incorporated at, butnot limited to, one or more of the following positions of FGF-21: 87,77, 83, 72, 69, 79, 91, 96, 108, and 110 of SEQ ID NO: 1 or thecorresponding amino acids in SEQ ID NOs: 2-7). In some embodiments, oneor more non-naturally encoded encoded amino acids are incorporated at,but not limited to, one or more of the following positions of FGF-21:87, 77, 83, 72 of SEQ ID NO: 1 or the corresponding amino acids in SEQID NOs: 2-7). In some embodiments, one or more non-naturally encodedencoded amino acids are incorporated at, but not limited to, one or moreof the following positions of FGF-21: 69, 79, 91, 96, 108, and 110 ofSEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs: 2-7).

The following criteria were used to evaluate each position of FGF-21 forthe introduction of a non-naturally encoded amino acid: the residue (a)should not interfere with binding of the FGF-21 Receptor based onstructural analysis, b) should not be affected by alanine or homologscanning mutagenesis (c) should be surface exposed and exhibit minimalvan der Waals or hydrogen bonding interactions with surroundingresidues, (d) should be either deleted or variable in FGF-21 variants,(e) would result in conservative changes upon substitution with anon-naturally encoded amino acid and (f) could be found in either highlyflexible regions or structurally rigid regions.

Additional or different crystal structures for members of the FGF familysuch as structures for FGF-23 and/or FGF-19 may also be used to selectsites for incorporation of one or more non-naturally encoded amino acidsinto FGF-21. For example, the crystal structure of human FGF-19 (PDB ID2P23) and/or crystal structure of human FGF-19 (PDB ID 2P23) and/orhuman FGF-23 (PDB ID 2P39) may provide additional information to selectsites for incorporation of non-naturally encoded amino acids intoFGF-21. Such sites may be in different regions of the protein, includingbut not limited to, the N- and C-termini, receptor binding and heparinbinding regions. In addition, further calculations can be performed onthe FGF-21 molecule, utilizing the Cx program (Pintar et al. (2002)Bioinformatics, 18, pp 980) to evaluate the extent of protrusion foreach protein atom.

In some embodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in FGF-21: beforeposition 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 (i.e., at thecarboxyl terminus of the protein) (SEQ ID NO: 1 or the correspondingamino acids in SEQ ID NOs: 2-7). In some embodiments, one or morenon-naturally encoded amino acids are incorporated in one or more of thefollowing positions in FGF-21: 10, 52, 117, 126, 131, 162, 87, 77, 83,72, 69, 79, 91, 96, 108, and 110 (SEQ ID NO: 1 or the correspondingamino acids of SEQ ID NOs: 2-7). In some embodiments, one or morenon-naturally encoded amino acids are incorporated in one or more of thefollowing positions in FGF-21: 10, 52, 77, 117, 126, 131, and 162 (SEQID NO: 1 or the corresponding amino acids of SEQ ID NOs: 2-7). In someembodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in FGF-21: 87,77, 83, 72 (SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs:2-7). In some embodiments, one or more non-naturally encoded amino acidsare incorporated in one or more of the following positions in FGF-21:69, 79, 91, 96, 108, and 110 (SEQ ID NO: 1 or the corresponding aminoacids of SEQ ID NOs: 2-7).

Example 2

This example details cloning and expression of a FGF-21 polypeptideincluding a non-naturally encoded amino acid in E. coli. This examplealso describes one method to assess the biological activity of modifiedFGF-21 polypeptides.

Methods for cloning FGF-21 are known to those of ordinary skill in theart. Polypeptide and polynucleotide sequences for FGF-21 and cloning ofFGF-21 into host cells are detailed in U.S. Pat. No. 6,716,626; U.S.Patent Publication Nos. 2005/0176631, 2005/0037457, 2004/0185494,2004/0259780, 2002/0164713, and 2001/0012628; WO 01/36640; WO 03/011213;WO 03/059270; WO 04/110472; WO 05/061712; WO 05/072769; WO 05/091944; WO05/113606; WO 06/028595; WO 06/028714; WO 06/050247; WO 06/065582; WO06/078463, which are incorporated by reference in their entirety herein.

cDNA encoding the P form of FGF-21 without the leader sequence is shownas SEQ ID NO: 8. This polypeptide is shown as SEQ ID NO: 1.

cDNA encoding a His tagged P form of FGF-21 without a leader sequence isshown as SEQ ID NO: 9. This polypeptide is shown as SEQ ID NO: 2.

cDNA encoding the P form of FGF-21 with a leader sequence containing 3leucines together is shown as SEQ ID NO: 10. This polypeptide is shownas SEQ ID NO: 3.

cDNA encoding the P form of FGF-21 with a leader sequence containing 2leucines together is shown as SEQ ID NO: 11. This polypeptide is shownas SEQ ID NO: 4.

cDNA encoding the L form of FGF-21 without the leader sequence is shownas SEQ ID NO: 12. This polypeptide is shown as SEQ ID NO: 5.

cDNA encoding the L form of FGF-21 with a leader sequence containing 3leucines together is shown as SEQ ID NO: 13. This polypeptide is shownas SEQ ID NO: 6.

cDNA encoding the L form of FGF-21 with a leader sequence containing 2leucines together is shown as SEQ ID NO: 14. This polypeptide is shownas SEQ ID NO: 7.

An introduced translation system that comprises an orthogonal tRNA(O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O—RS) is used toexpress FGF-21 containing a non-naturally encoded amino acid. The O—RSpreferentially aminoacylates the O-tRNA with a non-naturally encodedamino acid. In turn the translation system inserts the non-naturallyencoded amino acid into FGF-21, in response to an encoded selectorcodon. Suitable O—RS and O-tRNA sequences are described in WO2006/068802 entitled “Compositions of Aminoacyl-tRNA Synthetase and UsesThereof” (E9; SEQ ID NO: 15) and WO 2007/021297 entitled “Compositionsof tRNA and Uses Thereof” (F13; SEQ ID NO: 16), which are incorporatedby reference in their entirety herein.

TABLE 2 O-RS and O-tRNA sequences. SEQ ID NO: 17 M. jannaschiimtRNA_(CUA) ^(Tyr) tRNA SEQ ID NO: 18 HLAD03; an optimized ambersupressor tRNA tRNA SEQ ID NO: 19 HL325A; an optimized AGGA frameshiftsupressor tRNA tRNA SEQ ID NO: 20 Aminoacyl tRNA synthetase for theincorporation of p-azido-L-phenylalanine p-Az-PheRS(6) RS SEQ ID NO: 21Aminoacyl tRNA synthetase for the incorporation ofp-benzoyl-L-phenylalanine p-BpaRS(1) RS SEQ ID NO: 22 Aminoacyl tRNAsynthetase for the incorporation of propargyl-phenylalaninePropargyl-PheRS RS SEQ ID NO: 23 Aminoacyl tRNA synthetase for theincorporation of propargyl-phenylalanine Propargyl-PheRS RS SEQ ID NO:24 Aminoacyl tRNA synthetase for the incorporation ofpropargyl-phenylalanine Propargyl-PheRS RS SEQ ID NO: 25 Aminoacyl tRNAsynthetase for the incorporation of p-azido-phenylalanine p-Az-PheRS(1)RS SEQ ID NO: 26 Aminoacyl tRNA synthetase for the incorporation ofp-azido-phenylalanine p-Az-PheRS(3) RS SEQ ID NO: 27 Aminoacyl tRNAsynthetase for the incorporation of p-azido-phenylalanine p-Az-PheRS(4)RS SEQ ID NO: 28 Aminoacyl tRNA synthetase for the incorporation ofp-azido-phenylalanine p-Az-PheRS(2) RS SEQ ID NO: 29 Aminoacyl tRNAsynthetase for the incorporation of p-acetyl-phenylalanine (LW1) RS SEQID NO: 30 Aminoacyl tRNA synthetase for the incorporation of p-acetyl-phenylalanine (LW5) RS SEQ ID NO: 31 Aminoacyl tRNA synthetase for theincorporation of p-acetyl-phenylalanine (LW6) RS SEQ ID NO: 32 AminoacyltRNA synthetase for the incorporation of p-azido-phenylalanine(AzPheRS-5) RS SEQ ID NO: 33 Aminoacyl tRNA synthetase for theincorporation of p-azido-phenylalanine (AzPheRS-6) RS

The transformation of E. coli with plasmids containing the modifiedFGF-21 gene and the orthogonal aminoacyl tRNA synthetase/tRNA pair(specific for the desired non-naturally encoded amino acid) allows thesite-specific incorporation of non-naturally encoded amino acid into theFGF-21 polypeptide.

Wild type mature FGF-21 was amplified by PCR from a cDNA synthesisreaction derived from healthy human liver polyA+ mRNA (Biochain) usingstandard protocols and cloned into pET30 (Ncol-BamHI). Followingsequence confirmation, FGF-21 including an N-terminal HHHHHHSGG sequencewas subcloned into a suppression vector containing an amber suppressortyrosyl tRNA^(Tyr/CUA) from Methanococcus jannaschii (Mj tRNA^(Tyr/CUA))under constitutive control of a synthetic promoter derived from the E.coli lipoprotein promoter sequence (Miller, J. H., Gene, 1986), as wellas well as the orthogonal tyrosyl-tRNA-synthetase (MjTyrRS) undercontrol of the E. coli GlnRS promoter. Expression of FGF-21 was undercontrol of the T₇ promoter. Amber mutations were introduced usingstandard quick change mutation protocols (Stratagene; La Jolla, Calif.).All constructs were sequence verified.

Suppression with para-acetyl-phenylalanine (pAcF)

Plasmids (pVK3-HisFGF21) were transformed into the W3110 B2 strain of E.coli in which expression of the T₇ polymerase was under control of anarabinose-inducible promoter. Overnight bacterial cultures were diluted1:100 into shake flasks containing 2×YT culture media and grown at 37°C. to an OD₆₀₀ of ˜0.8. Protein expression was induced by the additionof arabinose (0.2% final), and para-acetyl-phenylalanine (pAcF) to afinal concentration of 4 mM. Cultures were incubated at 37° C. for 4hours. Cells were pelleted and resuspended in B-PER lysis buffer(Pierce) 100 ul/OD/ml+10 ug/ml DNase and incubated at 37° C. for 30 min.Cellular material was removed by centrifugation and the supernatantremoved. The pellet was re-suspended in an equal amount of SDS-PAGEprotein loading buffer. All samples were loaded on a 4-12% PAGE gel withIVIES and DTT. Methods for purification of FGF-21 are known to those ofordinary skill in the art and are confirmed by SDS-PAGE, Western Blotanalyses, or electrospray-ionization ion trap mass spectrometry and thelike.

Expression of N-terminal His tagged FGF-21 and suppression at 7 ambersites is shown as FIG. 5. The FGF-21 polypeptide is marked with anarrow. FIG. 5 shows the B-PER pellet samples—Lane 1: Marker; Lane 2:VK3-FGF21 preinduction, supernatant; Lane 3: VK3-FGF21 preinduction,pellet; Lane 4: VK3-FGF21 0.2% arabinose, supernatant; Lane 5: VK3-FGF210.2% arabinose, pellet; Lane 6: VK3-FGF21-pAcF-L10, 0.2% arabinose; Lane7: VK3-FGF21-pAcF-L52, 0.2% arabinose; Lane 8: VK3-FGF21-pAcF-R77, 0.2%arabinose; Lane 9: VK3-FGF21-pAcF-H117, 0.2% arabinose; Lane 10:VK3-FGF21-pAcF-R126, 0.2% arabinose; Lane 11: VK3-FGF21-pAcF-R131, 0.2%arabinose; Lane 12: VK3-FGF21-pAcF-5162, 0.2% arabinose. The positionnumbers indicated for the amino acid substitution are based on SEQ IDNO: 1.

FIG. 6 shows the B-PER supernatant samples—Lane 1: VK3-FGF21preinduction, supernatant; Lane 2: VK3-FGF21 preinduction, pellet; Lane3: Marker; Lane 4: VK3-FGF21 0.2% arabinose, supernatant; Lane 5:VK3-FGF21 0.2% arabinose, pellet; Lane 6: VK3-FGF21-pAcF-L10, 0.2%arabinose; Lane 7: VK3-FGF21-pAcF-L52, 0.2% arabinose; Lane 8:VK3-FGF21-pAcF-R77, 0.2% arabinose; Lane 9: VK3-FGF21-pAcF-H117, 0.2%arabinose; Lane 10: VK3-FGF21-pAcF-R126, 0.2% arabinose; Lane 11:VK3-FGF21-pAcF-R131, 0.2% arabinose; Lane 12: VK3-FGF21-pAcF-5162, 0.2%arabinose. The position numbers indicated for the amino acidsubstitution are based on SEQ ID NO: 1.

His-tagged mutant FGF-21 proteins can be purified using methods known tothose of ordinary skill in the art. The ProBond Nickel-Chelating Resin(Invitrogen, Carlsbad, Calif.) may be used via the standard His-taggedprotein purification procedures provided by the manufacturer.

pVK10 (FIG. 24) was developed for use with the untagged FGF-21 protein,having a sequence given in FIG. 25. This was the vector used to make R₃₆am and Y83 am mutants and there is further data on this non-His taggedFGF-21 mutant proteins and their purification later in the examples andshown throughout the figures.

Differentiation of 3T₃-L1 to adipocytes and glucose uptake assay

To assess the biological activity of FGF-21 polypeptides, the followingassay may be performed. Mouse 3T₃-L1 fibroblasts (ATCC #CL-173) areseeded in a 10 cm dish with DMEM containing 10% bovine calf serum. Thecells are kept at a density not higher the 70% for expansion. Beforestarting differentiation to adipocytes, the cells are allowed to go to100% confluence; the medium has to be changed every 2 days. The cellsare counted and seeded at 25,000cells/well in a 96 well/plate (cells canalso be plated on Cytostar-T 96 well/plate) and incubated for another 48hours. Differentiation is induced by adding the following medium afterremoving the previous culture medium: DMEM supplemented with 10% FBS(Fetal Calf Serum), 1 μM dexamethasone (DBX), 0.5 mM3-isobutyl-1-methylxanthine (IBMX), and 5 μg/mL insulin. An alternativeway to induce differentiation is to treat the cells with 1 μMRosiglitazone and incubate for 6 days before changing medium to DMEM/10%FBS, since this is a faster way to induce 3T₃-L1 fibroblasts todifferentiate into adipocytes. A third possibility is to combine the twoprocedures to shorten time for differentiation.

After adding DBX/IBMX/insulin containing medium to cells, the cells areincubated for 48 hours. The medium is changed to DMEM/10% FBS/5 μg/mLinsulin, and the cells are incubated for 48 hours. Thereafter the mediumis changed to DMEM/10% FBS and the medium is replaced with fresh mediumevery 2 days. The cells will differentiate between 7-14 days.Differentiated cells accumulate lipid droplets. The cells can be stainedwith OIL RED O. Once the 95% adipocytes contain lipid droplets, thecells can be used for the glucose uptake assay.

Differentiated 3T₃-L1 are treated with FGF-21 (1 μg/mL) in DMEMsupplemented with 0.1% fatty acid free-BSA for 18 hours to starve thecells. The cells are then washed 3 times with Kreb's-Ringer HEPES buffer(KRH=0.118M NaCl, 5 mM KCl, 2.54 mM CaCl₂), 1.19 mM KH₂PO₄, 1.19 mMMgSO₄, and 20 mM HEPES) supplemented with 0.1% FAF-BSA. The labeling mixis prepared by adding 4 μCi, 0.1 mM of 2-deoxyD-[1-³H]-glucose toKRH/0.1% FAF-BSA buffer. The cells are added and incubated for 1 hour at37° C. The reaction is stopped by washing the cells twice with ice-coldPBS containing 20 μM cytochalasin B. The plate is blotted to eliminateany residual buffer. Scintillitaion liquid is added to each well andsamples are counted on a TopCounter.

An alternative way to measure glucose uptake is to load thedifferentiated 3T₃-L1 cells with a non-radioactive substrate as 2-NBDGand read with a fluorescence plate reader. An indirect procedure formeasuring glucose uptake is to measure the expression GLUT1 or GLUT4 onthe cell membrane surface. GLUT1 and GLUT4 are glucose transporters thatare translocated on the cell membrane surface from the internal vesiclesupon insulin or FGF-21 stimulation. An ELISA on live cells can bedeveloped.

Example 3

This example details introduction of a carbonyl-containing amino acidand subsequent reaction with an aminooxy-containing PEG.

This Example demonstrates a method for the generation of a FGF-21polypeptide that incorporates a ketone-containing non-naturally encodedamino acid that is subsequently reacted with an aminooxy-containing PEGof approximately 5,000 MW. Each of the residues before position 1 (i.e.at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182 (i.e., at the carboxyl terminusof the protein) (SEQ ID NO: 1 or the corresponding amino acids in SEQ IDNOs: 2-7) is separately substituted with a non-naturally encoded aminoacid having the following structure:

The sequences utilized for site-specific incorporation ofp-acetyl-phenylalanine into FGF-21 are SEQ ID NO: 1 (FGF-21), and SEQ IDNO: 16 or 17 (muttRNA, M. jannaschii mtRNA_(CUA) ^(Tyr)), and 15, 29, 30or 31 (TyrRS LW1, 5, or 6) described in Example 2 above.

Once modified, the FGF-21 polypeptide variant comprising thecarbonyl-containing amino acid is reacted with an aminooxy-containingPEG derivative of the form:

R-PEG(N)—O—(CH₂)_(n)—O—NH₂

where R is methyl, n is 3 and N is approximately 5,000 MW. The purifiedFGF-21 containing p-acetylphenylalanine dissolved at 10 mg/mL in 25 mMIVIES (Sigma Chemical, St. Louis, Mo.) pH 6.0, 25 mM Hepes (SigmaChemical, St. Louis, Mo.) pH 7.0, or in 10 mM Sodium Acetate (SigmaChemical, St. Louis, Mo.) pH 4.5, is reacted with a 10 to 100-foldexcess of aminooxy-containing PEG, and then stirred for 10-16 hours atroom temperature (Jencks, W. J. Am. Chem. Soc. 1959, 81, pp 475). ThePEG-FGF-21 is then diluted into appropriate buffer for immediatepurification and analysis.

Example 4

Conjugation with a PEG consisting of a hydroxylamine group linked to thePEG via an amide linkage.

A PEG reagent having the following structure is coupled to aketone-containing non-naturally encoded amino acid using the proceduredescribed in Example 3:

R-PEG(N)—O—(CH₂)₂—NH—C(O)(CH₂)_(n)—O—NH₂

where R=methyl, n=4 and N is approximately 20,000 MW. The reaction,purification, and analysis conditions are as described in Example 3.

Example 5

This example details the introduction of two distinct non-naturallyencoded amino acids into FGF-21 polypeptides.

This example demonstrates a method for the generation of a FGF-21polypeptide that incorporates non-naturally encoded amino acidcomprising a ketone functionality at two positions among the followingresidues: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182(i.e., at the carboxyl terminus of the protein) (SEQ ID NO: 1 or thecorresponding amino acids in SEQ ID NOs: 2-7). The FGF-21 polypeptide isprepared as described in Examples 1 and 2, except that the selectorcodon is introduced at two distinct sites within the nucleic acid.

Example 6

This example details conjugation of FGF-21 polypeptide to ahydrazide-containing PEG and subsequent in situ reduction.

A FGF-21 polypeptide incorporating a carbonyl-containing amino acid isprepared according to the procedure described in Examples 2 and 3. Oncemodified, a hydrazide-containing PEG having the following structure isconjugated to the FGF-21 polypeptide:

R-PEG(N)—O—(CH₂)₂—NH—C(O)(CH₂)_(n)—X—NH—NH₂

where R=methyl, n=2 and N=10,000 MW and X is a carbonyl (C═O) group. Thepurified FGF-21 containing p-acetylphenylalanine is dissolved at between0.1-10 mg/mL in 25 mM MES (Sigma Chemical, St. Louis, Mo.) pH 6.0, 25 mMHepes (Sigma Chemical, St. Louis, Mo.) pH 7.0, or in 10 mM SodiumAcetate (Sigma Chemical, St. Louis, Mo.) pH 4.5, is reacted with a 1 to100-fold excess of hydrazide-containing PEG, and the correspondinghydrazone is reduced in situ by addition of stock 1M NaCNBH3 (SigmaChemical, St. Louis, Mo.), dissolved in H₂O, to a final concentration of10-50 mM. Reactions are carried out in the dark at 4° C. to RT for 18-24hours. Reactions are stopped by addition of 1 M Tris (Sigma Chemical,St. Louis, Mo.) at about pH 7.6 to a final Tris concentration of 50 mMor diluted into appropriate buffer for immediate purification.

Example 7

This example details introduction of an alkyne-containing amino acidinto a FGF-21 polypeptide and derivatization with mPEG-azide.

The following residues, before position 1 (i.e. at the N-terminus), 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182 (i.e., at the carboxyl terminus of the protein) (SEQID NO: 1 or the corresponding amino acids in SEQ ID NOs: 2-7), are eachsubstituted with the following non-naturally encoded amino acid:

The sequences utilized for site-specific incorporation ofp-propargyl-tyrosine into FGF-21 are SEQ ID NO: 1 (FGF-21), SEQ ID NO:16 or 17 (muttRNA, M jannaschii mtRNA_(CUA) ^(Tyr)), and 22, 23 or 24described in Example 2 above. The FGF-21 polypeptide containing thepropargyl tyrosine is expressed in E. coli and purified using theconditions described in Example 3.

The purified FGF-21 containing propargyl-tyrosine dissolved at between0.1-10 mg/mL in PB buffer (100 mM sodium phosphate, 0.15 M NaCl, pH=8)and a 10 to 1000-fold excess of an azide-containing PEG is added to thereaction mixture. A catalytic amount of CuSO₄ and Cu wire are then addedto the reaction mixture. After the mixture is incubated (including butnot limited to, about 4 hours at room temperature or 37° C., orovernight at 4° C.), H₂O is added and the mixture is filtered through adialysis membrane. The sample can be analyzed for the addition,including but not limited to, by similar procedures described in Example3.

In this Example, the PEG will have the following structure:

R-PEG(N)—O—(CH₂)₂—NH—C(O)(CH₂)_(n)—N₃

where R is methyl, n is 4 and N is 10,000 MW.

Example 8

This example details substitution of a large, hydrophobic amino acid ina FGF-21 polypeptide with propargyl tyrosine.

A Phe, Trp or Tyr residue present within one the following regions ofFGF-21: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182(i.e., at the carboxyl terminus of the protein) (SEQ ID NO: 1 or thecorresponding amino acids in SEQ ID NOs: 2-7) is substituted with thefollowing non-naturally encoded amino acid as described in Example 7:

Once modified, a PEG is attached to the FGF-21 polypeptide variantcomprising the alkyne-containing amino acid. The PEG will have thefollowing structure:

Me-PEG(N)—O—(CH₂)₂—N₃

and coupling procedures would follow those in Example 7. This willgenerate a FGF-21 polypeptide variant comprising a non-naturally encodedamino acid that is approximately isosteric with one of thenaturally-occurring, large hydrophobic amino acids and which is modifiedwith a PEG derivative at a distinct site within the polypeptide.

Example 9

This example details generation of a FGF-21 polypeptide homodimer,heterodimer, homomultimer, or heteromultimer separated by one or morePEG linkers.

The alkyne-containing FGF-21 polypeptide variant produced in Example 7is reacted with a bifunctional PEG derivative of the form:

N₃—(CH₂)_(n)—C(O)—NH—(CH₂)₂—O-PEG(N)—O—(CH₂)₂—NH—C(O)—(CH₂)_(n)—N₃

where n is 4 and the PEG has an average MW of approximately 5,000, togenerate the corresponding FGF-21 polypeptide homodimer where the twoFGF-21 molecules are physically separated by PEG. In an analogous mannera FGF-21 polypeptide may be coupled to one or more other polypeptides toform heterodimers, homomultimers, or heteromultimers. Coupling,purification, and analyses will be performed as in Examples 7 and 3.

Example 10

This example details coupling of a saccharide moiety to a FGF-21polypeptide.

One residue of the following is substituted with the non-naturallyencoded amino acid below: before position 1 (i.e. at the N-terminus), 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182 (i.e., at the carboxyl terminus of the protein) (SEQID NO: 1 or the corresponding amino acids in SEQ ID NOs: 2-7) asdescribed in Example 3.

Once modified, the FGF-21 polypeptide variant comprising thecarbonyl-containing amino acid is reacted with a β-linked aminooxyanalogue of N-acetylglucosamine (GlcNAc). The FGF-21 polypeptide variant(10 mg/mL) and the aminooxy saccharide (21 mM) are mixed in aqueous 100mM sodium acetate buffer (pH 5.5) and incubated at 37° C. for 7 to 26hours. A second saccharide is coupled to the first enzymatically byincubating the saccharide-conjugated FGF-21 polypeptide (5 mg/mL) withUDP-galactose (16 mM) and β-1,4-galacytosyltransferase (0.4 units/mL) in150 mM HEPES buffer (pH 7.4) for 48 hours at ambient temperature(Schanbacher et al. J. Biol. Chem. 1970, 245, 5057-5061).

Example 11

This example details generation of a PEGylated FGF-21 polypeptideantagonist.

A residue, including but not limited to, those involved in FGF-21receptor binding is substituted with the following non-naturally encodedamino acid as described in Example 3.

Once modified, the FGF-21 polypeptide variant comprising thecarbonyl-containing amino acid will be reacted with anaminooxy-containing PEG derivative of the form:

R-PEG(N)—O—(CH₂)_(n)—O—NH₂

where R is methyl, n is 4 and N is 20,000 MW to generate a FGF-21polypeptide antagonist comprising a non-naturally encoded amino acidthat is modified with a PEG derivative at a single site within thepolypeptide. Coupling, purification, and analyses are performed as inExample 3.

Example 12

Generation of a FGF-21 polypeptide homodimer, heterodimer, homomultimer,or heteromultimer in which the FGF-21 Molecules are Linked Directly

A FGF-21 polypeptide variant comprising the alkyne-containing amino acidcan be directly coupled to another FGF-21 polypeptide variant comprisingthe azido-containing amino acid. In an analogous manner a FGF-21polypeptide polypeptide may be coupled to one or more other polypeptidesto form heterodimers, homomultimers, or heteromultimers. Coupling,purification, and analyses are performed as in Examples 3, 6, and 7.

Example 13

The polyalkylene glycol (P—OH) is reacted with the alkyl halide (A) toform the ether (B). In these compounds, n is an integer from one to nineand R′ can be a straight- or branched-chain, saturated or unsaturatedC1, to C20 alkyl or heteroalkyl group. R′ can also be a C3 to C7saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, asubstituted or unsubstituted aryl or heteroaryl group, or a substitutedor unsubstituted alkaryl (the alkyl is a C1 to C20 saturated orunsaturated alkyl) or heteroalkaryl group. Typically, PEG-OH ispolyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG)having a molecular weight of 800 to 40,000 Daltons (Da).

Example 14

mPEG-OH+Br-CH₂—C≡CH→mPEG-O—CH₂—C≡CH

mPEG-OH with a molecular weight of 20,000 Da (mPEG-OH 20 kDa; 2.0 g, 0.1mmol, Sunbio) was treated with NaH (12 mg, 0.5 mmol) in THF (35 mL). Asolution of propargyl bromide, dissolved as an 80% weight solution inxylene (0.56 mL, 5 mmol, 50 equiv., Aldrich), and a catalytic amount ofKI were then added to the solution and the resulting mixture was heatedto reflux for 2 hours. Water (1 mL) was then added and the solvent wasremoved under vacuum. To the residue was added CH₂Cl₂ (25 mL) and theorganic layer was separated, dried over anhydrous Na₂SO₄, and the volumewas reduced to approximately 2 mL. This CH₂Cl₂ solution was added todiethyl ether (150 mL) drop-wise. The resulting precipitate wascollected, washed with several portions of cold diethyl ether, and driedto afford propargyl-O-PEG.

Example 15

mPEG-OH+Br-(CH₂)₃—C≡CH→mPEG-O—(CH₂)₃—C≡CH

The mPEG-OH with a molecular weight of 20,000 Da (mPEG-OH 20 kDa; 2.0 g,0.1 mmol, Sunbio) was treated with NaH (12 mg, 0.5 mmol) in THF (35 mL).Fifty equivalents of 5-bromo-1-pentyne (0.53 mL, 5 mmol, Aldrich) and acatalytic amount of KI were then added to the mixture. The resultingmixture was heated to reflux for 16 hours. Water (1 mL) was then addedand the solvent was removed under vacuum. To the residue was addedCH₂Cl₂ (25 mL) and the organic layer was separated, dried over anhydrousNa₂SO₄, and the volume was reduced to approximately 2 mL. This CH₂Cl₂solution was added to diethyl ether (150 mL) drop-wise. The resultingprecipitate was collected, washed with several portions of cold diethylether, and dried to afford the corresponding alkyne. 5-chloro-1-pentynemay be used in a similar reaction.

Example 16

m-HOCH₂C₆H₄OH+NaOH+Br—CH₂—C≡CH→m-HOCH₂C₆H₄O—CH₂—C≡CH  (1)

m-HOCH2C₆H₄O—CH₂—C≡CH+MsCl+N(Et)₃→m-MsOCH₂C₆H₄O—CH₂—C≡CH  (2)

m-MsOCH₂C₆H₄O—CH₂—C≡CH+LiBr→m-Br—CH₂C₆H₄O—CH₂—C≡CH  (3)

mPEG-OH+m-Br—CH₂C₆H₄O—CH₂—C≡CH→mPEG-O—CH₂—C₆H₄O—CH₂—C≡CH  (4)

To a solution of 3-hydroxybenzylalcohol (2.4 g, 20 mmol) in THF (50 mL)and water (2.5 mL) was first added powdered sodium hydroxide (1.5 g,37.5 mmol) and then a solution of propargyl bromide, dissolved as an 80%weight solution in xylene (3.36 mL, 30 mmol). The reaction mixture washeated at reflux for 6 hours. To the mixture was added 10% citric acid(2.5 mL) and the solvent was removed under vacuum. The residue wasextracted with ethyl acetate (3×15 mL) and the combined organic layerswere washed with saturated NaCl solution (10 mL), dried over MgSO₄ andconcentrated to give the 3-propargyloxybenzyl alcohol.

Methanesulfonyl chloride (2.5 g, 15.7 mmol) and triethylamine (2.8 mL,20 mmol) were added to a solution of compound 3 (2.0 g, 11.0 mmol) inCH₂Cl₂ at 0° C. and the reaction was placed in the refrigerator for 16hours. A usual work-up afforded the mesylate as a pale yellow oil. Thisoil (2.4 g, 9.2 mmol) was dissolved in THF (20 mL) and LiBr (2.0 g, 23.0mmol) was added. The reaction mixture was heated to reflux for 1 hourand was then cooled to room temperature. To the mixture was added water(2.5 mL) and the solvent was removed under vacuum. The residue wasextracted with ethyl acetate (3×15 mL) and the combined organic layerswere washed with saturated NaCl solution (10 mL), dried over anhydrousNa₂SO₄, and concentrated to give the desired bromide.

mPEG-OH 20 kDa (1.0 g, 0.05 mmol, Sunbio) was dissolved in THF (20 mL)and the solution was cooled in an ice bath. NaH (6 mg, 0.25 mmol) wasadded with vigorous stirring over a period of several minutes followedby addition of the bromide obtained from above (2.55 g, 11.4 mmol) and acatalytic amount of KI. The cooling bath was removed and the resultingmixture was heated to reflux for 12 hours. Water (1.0 mL) was added tothe mixture and the solvent was removed under vacuum. To the residue wasadded CH₂Cl₂ (25 mL) and the organic layer was separated, dried overanhydrous Na₂SO₄, and the volume was reduced to approximately 2 mL.Dropwise addition to an ether solution (150 mL) resulted in a whiteprecipitate, which was collected to yield the PEG derivative.

Example 17

mPEG-NH₂+X—C(O)—(CH₂)_(n)—C≡CR′→mPEG-NH—C(O)—(CH₂)_(n)—C≡CR′

The terminal alkyne-containing poly(ethylene glycol) polymers can alsobe obtained by coupling a poly(ethylene glycol) polymer containing aterminal functional group to a reactive molecule containing the alkynefunctionality as shown above. n is between 1 and 10. R′ can be H or asmall alkyl group from C1 to C4.

Example 18

HO₂C—(CH₂)₂—C≡CH+NHS+DCC→NHSO—C(O)—(CH₂)₂—C≡CH  (1)

mPEG-NH₂+NHSO—C(O)—(CH₂)₂—C≡CH→mPEG-NEI—C(O)—(CH₂)₂—C≡CH  (2)

4-pentynoic acid (2.943 g, 3.0 mmol) was dissolved in CH₂Cl₂ (25 mL).N-hydroxysuccinimide (3.80 g, 3.3 mmol) and DCC (4.66 g, 3.0 mmol) wereadded and the solution was stirred overnight at room temperature. Theresulting crude NHS ester 7 was used in the following reaction withoutfurther purification.

mPEG-NH₂ with a molecular weight of 5,000 Da (mPEG-NH₂, 1 g, Sunbio) wasdissolved in THF (50 mL) and the mixture was cooled to 4° C. NHS ester 7(400 mg, 0.4 mmol) was added portion-wise with vigorous stirring. Themixture was allowed to stir for 3 hours while warming to roomtemperature. Water (2 mL) was then added and the solvent was removedunder vacuum. To the residue was added CH₂Cl₂ (50 mL) and the organiclayer was separated, dried over anhydrous Na₂SO₄, and the volume wasreduced to approximately 2 mL. This CH₂Cl₂ solution was added to ether(150 mL) drop-wise. The resulting precipitate was collected and dried invacuo.

Example 19

This Example represents the preparation of the methane sulfonyl ester ofpoly(ethylene glycol), which can also be referred to as themethanesulfonate or mesylate of poly(ethylene glycol). The correspondingtosylate and the halides can be prepared by similar procedures.

mPEG-OH+CH₃SO₂Cl+N(Et)₃→mPEG-O—SO₂CH₃→mPEG-N₃

The mPEG-OH (MW=3,400, 25 g, 10 mmol) in 150 mL of toluene wasazeotropically distilled for 2 hours under nitrogen and the solution wascooled to room temperature. 40 mL of dry CH₂Cl₂ and 2.1 mL of drytriethylamine (15 mmol) were added to the solution. The solution wascooled in an ice bath and 1.2 mL of distilled methanesulfonyl chloride(15 mmol) was added dropwise. The solution was stirred at roomtemperature under nitrogen overnight, and the reaction was quenched byadding 2 mL of absolute ethanol. The mixture was evaporated under vacuumto remove solvents, primarily those other than toluene, filtered,concentrated again under vacuum, and then precipitated into 100 mL ofdiethyl ether. The filtrate was washed with several portions of colddiethyl ether and dried in vacuo to afford the mesylate.

The mesylate (20 g, 8 mmol) was dissolved in 75 ml of THF and thesolution was cooled to 4° C. To the cooled solution was added sodiumazide (1.56 g, 24 mmol). The reaction was heated to reflux undernitrogen for 2 hours. The solvents were then evaporated and the residuediluted with CH₂Cl₂ (50 mL). The organic fraction was washed with NaClsolution and dried over anhydrous MgSO₄. The volume was reduced to 20 mland the product was precipitated by addition to 150 ml of cold dryether.

Example 20

N₃—C₆H₄—CO₂H→N₃—C₆H₄CH₂OH  (1)

N₃—C₆H₄CH₂OH→Br—CH₂—C₆H₄—N₃  (2)

mPEG-OH+Br—CH₂—C₆H₄—N₃→mPEG-O—CH₂—C₆H₄—N₃  (3)

4-azidobenzyl alcohol can be produced using the method described in U.S.Pat. No. 5,998,595, which is incorporated by reference herein.Methanesulfonyl chloride (2.5 g, 15.7 mmol) and triethylamine (2.8 mL,20 mmol) were added to a solution of 4-azidobenzyl alcohol (1.75 g, 11.0mmol) in CH₂Cl₂ at 0° C. and the reaction was placed in the refrigeratorfor 16 hours. A usual work-up afforded the mesylate as a pale yellowoil. This oil (9.2 mmol) was dissolved in THF (20 mL) and LiBr (2.0 g,23.0 mmol) was added. The reaction mixture was heated to reflux for 1hour and was then cooled to room temperature. To the mixture was addedwater (2.5 mL) and the solvent was removed under vacuum. The residue wasextracted with ethyl acetate (3×15 mL) and the combined organic layerswere washed with saturated NaCl solution (10 mL), dried over anhydrousNa₂SO₄, and concentrated to give the desired bromide.

mPEG-OH 20 kDa (2.0 g, 0.1 mmol, Sunbio) was treated with NaH (12 mg,0.5 mmol) in THF (35 mL) and the bromide (3.32 g, 15 mmol) was added tothe mixture along with a catalytic amount of KI. The resulting mixturewas heated to reflux for 12 hours. Water (1.0 mL) was added to themixture and the solvent was removed under vacuum. To the residue wasadded CH₂Cl₂ (25 mL) and the organic layer was separated, dried overanhydrous Na₂SO₄, and the volume was reduced to approximately 2 mL.Dropwise addition to an ether solution (150 mL) resulted in aprecipitate, which was collected to yield mPEG-O—CH₂—C₆H₄—N₃.

Example 21

NH₂-PEG-O-CH₂CH₂CO₂H+N₃—CH₂CH₂CO₂—NHS→N₃—CH₂CH₂—C(O)NH-PEG-O—CH₂CH₂CO₂H

NH₂-PEG-O—CH₂CH₂CO₂H (MW 3,400 Da, 2.0 g) was dissolved in a saturatedaqueous solution of NaHCO₃(10 mL) and the solution was cooled to 0° C.3-azido-1-N-hydroxysuccinimido propionate (5 equiv.) was added withvigorous stirring. After 3 hours, 20 mL of H₂O was added and the mixturewas stirred for an additional 45 minutes at room temperature. The pH wasadjusted to 3 with 0.5 N H₂SO₄ and NaCl was added to a concentration ofapproximately 15 wt %. The reaction mixture was extracted with CH₂Cl₂(100 mL×3), dried over Na₂SO₄ and concentrated. After precipitation withcold diethyl ether, the product was collected by filtration and driedunder vacuum to yield the omega-carboxy-azide PEG derivative.

Example 22

mPEG-OMs+HC≡CLi→mPEG-O—CH₂—CH₂—C≡C—H

To a solution of lithium acetylide (4 equiv.), prepared as known in theart and cooled to −78° C. in THF, is added dropwise a solution ofmPEG-OMs dissolved in THF with vigorous stirring. After 3 hours, thereaction is permitted to warm to room temperature and quenched with theaddition of 1 mL of butanol. 20 mL of H₂O is then added and the mixturewas stirred for an additional 45 minutes at room temperature. The pH wasadjusted to 3 with 0.5 N H₂SO₄ and NaCl was added to a concentration ofapproximately 15 wt %. The reaction mixture was extracted with CH₂Cl₂(100 mL×3), dried over Na₂SO₄ and concentrated. After precipitation withcold diethyl ether, the product was collected by filtration and driedunder vacuum to yield the 1-(but-3-ynyloxy)-methoxypolyethylene glycol(mPEG).

Example 23

Azide- and acetylene-containing amino acids can be incorporatedsite-selectively into proteins using the methods described in L. Wang,et al., (2001), Science 292:498-500, J.W. Chin et al., Science 301:964-7(2003)), J. W. Chin et al., (2002), Journal of the American ChemicalSociety 124:9026-9027; J. W. Chin, & P. G. Schultz, (2002), Chem BioChem 3(11):1135-1137; J. W. Chin, et al., (2002), PNAS United States ofAmerica 99:11020-11024: and, L. Wang, & P. G. Schultz, (2002), Chem.Comm., 1:1-11. Once the amino acids were incorporated, the cycloadditionreaction is carried out with 0.01 mM protein in phosphate buffer (PB),pH 8, in the presence of 2 mM PEG derivative, 1 mM CuSO₄, and −1 mgCu-wire for 4 hours at 37° C.

Example 24

This example describes the synthesis of p-Acetyl-D,L-phenylalanine (pAF)and m-PEG-hydroxylamine derivatives.

The racemic pAF is synthesized using the previously described procedurein Zhang, Z., Smith, B. A. C., Wang, L., Brock, A., Cho, C. & Schultz,P. G., Biochemistry, (2003) 42, 6735-6746.

To synthesize the m-PEG-hydroxylamine derivative, the followingprocedures are completed. To a solution of (N-t-Boc-aminooxy)acetic acid(0.382 g, 2.0 mmol) and 1,3-Diisopropylcarbodiimide (0.16 mL, 1.0 mmol)in dichloromethane (DCM, 70 mL), which is stirred at room temperature(RT) for 1 hour, methoxy-polyethylene glycol amine (m-PEG-NH₂, 7.5 g,0.25 mmol, Mt. 30 K, from BioVectra) and Diisopropylethylamine (0.1 mL,0.5 mmol) is added. The reaction is stirred at RT for 48 hours, and thenis concentrated to about 100 mL. The mixture is added dropwise to coldether (800 mL). The t-Boc-protected product precipitated out and iscollected by filtering, washed by ether 3×100 mL. It is further purifiedby re-dissolving in DCM (100 mL) and precipitating in ether (800 mL)twice. The product is dried in vacuum yielding 7.2 g (96%), confirmed byNMR and Nihydrin test.

The deBoc of the protected product (7.0 g) obtained above is carried outin 50% TFA/DCM (40 mL) at 0° C. for 1 hour and then at RT for 1.5 hour.After removing most of TFA in vacuum, the TFA salt of the hydroxylaminederivative is converted to the HCl salt by adding 4N HCl in dioxane (1mL) to the residue. The precipitate is dissolved in DCM (50 mL) andre-precipitated in ether (800 mL). The final product (6.8 g, 97%) iscollected by filtering, washed with ether 3×100 mL, dried in vacuum,stored under nitrogen. Other PEG (5K, 20K) hydroxylamine derivatives aresynthesized using the same procedure.

Example 25

Analysis of ERK1/2 phosphorylation induced by FGF-21 WT and 30K PEGanalogs:

Seed 293-stably transfected with human Klotho beta at 100,000 cells/well(DMEM+10% FBS) in a poly-Lys coated plate. The following day cells are100% confluent, media is aspirated off and replaced with fresh media andincubate overnight. After 24 hours cells are stimulated with theappropriate 30K PEG FGF-21 analogs using as standard FGF21WT. Eachindividual compound is prepared by diluting them in PBS/1% BSA. Cellsare treated in triplicate for 10 min @37° C. in the incubator. After 10min incubation media is carefully aspirated off from each well and 40 ulof cold 1× Cell Signaling Lysis Buffer containing protease/phosphataseinhibitors (PI cocktail, Na3VN4 and PMSF) are added to each well toproduce cell lysates. 96well/plate is placed on ice for 20 minutes andthen spun down at 4000 rpm for 10 min. Cell lysates are frozen down@-80° C. Later on each sample is thawed out and 10 ul of cell lysates isadded to MSD treated plate coated with antibody capturing both theunphosphorylated and phosphorylated forms of ERK1/2. Incubation withprimary antibody occurs for 2 hrs, then plate is washed several timeswith specific buffer followed by addition of secondary anitbody. After 1hour incubation plate is washed again several times. Buffer for readingis added to each well. Plate is transferred to MSD reading machine. Thecurve that is produced is based on the anti-phosphorylated ERK1/2reading units and EC50 is calculated using Sigma Plot. The fold loss ofactivity is calculated by dividing EC50 of the 30 K pegylated specificcompound with the EC50 of the WT.

Example 26: Cellular ERK 1/2 Phosphorylation Assay (pERK) Protocol andMSD Analysis

293 βKlotho-4 cells were maintained in DMEM+10% FBS+P/S+0.5 mg/mLGeneticin. When the cells reached 50-90% confluency, they weretrypsinized, and seeded 100,000 cells/well in poly-D-lys coated 96-wellplates in DMEM+10% FBS+P/S.

The following day when the cells were ˜100% confluent, they were checkedto be sure that the media was still red, then the media was aspiratedoff 200 uL/well of serial dilutions of FGF-21 variants (in PBS+1% BSA)were pipetted into the 96-well plate. The 96-well plate was then placedin 37° C., 5% CO₂ incubator for exactly 9 minutes. The FGF-21 treatmentswere then completely aspirated off and 40 uL/well of freshly made 1×CellSignaling Lysis Buffer+1× Sigma Protease Inhibitor Cocktail+2 mM SodiumOrthovanadate+1 mM PMSF+1×MSD Phosphatase Inhibitor I+1×MSD PhosphataseInhibitor II+1×MSD Protease Inhibitor Cocktail+2 mM MSD PMSF was added.The dish was placed on ice, and set aside for 25 minutes, while, pipetteeach well up and down with a P20 pipettor to mix the lysates. Aftermixing wells, place the whole ice bucket and dish on 4C shaker for theremainder of the time. After 25 minutes, spin down the plate at 4000rpm, 10 min, at 4C. Transfer the supernatants to a cold round-bottom96-well plate on ice.

MSD Analysis

This assay was performed using the Meso Scale Discovery MULTI-SPOT AssaySystem whole-cell lysate kit Phospho (T/Y 202/204; 185/187)/Total—ERK1/2/Assay.

All MSD reagents were thawed to room temperature and all necessarybuffers were made per kit instructions. To each well, 150 uL of BlockerA was added to a phosphoERK-totalERK Duplex plate and it was allowed toblock for 1 hr at room temperature on a shaker. The plate was thenwashed 4× with 160 uL/well of 1× Wash Buffer. 16 uL/well of LysisBuffer+protease and phosphatase inhibitors (made earlier for the cellstimulations) were added and 10 uL/well were transferred from the coldlysate supernatant plate to the MSD plate wells (total volume thenbecame 26 uL/well). The MSD plate incubated for 3 hrs at roomtemperature on a shaker the plate was then washed 4× with 160 uL/well of1× Wash Buffer. 25 uL/well of Detection Antibody (diluted 50× inAntibody Dilution Buffer) were added and set to incubate for 1 hr atroom temperature on a shaker. The plate was again washed 4× with 160uL/well of 1× Wash Buffer and 150 uL/well of 1× Read Buffer T was addedafter which the plate was immediately read on MSD Sector Imager 2400machine.

BCA Quantification

The Pierce BCA Protein Assay Kit was used. BSA standard was diluted in a96-well plate from a top concentration of 2 mg/mL, with 2× dilutionsdown the columns in 1×Cell Signaling Lysis Buffer. (last set of wellswere buffer, no BSA) 25 uL/well of the BSA standards were pipetted induplicate to two MaxiSorp 96-well plates. 3× dilutions of lysates weremade in 1×Cell Signaling Lysis Buffer and 25 uL/well were added to theMaxiSorp plates. The Working Reagent was made as per the Pierce Kitinstruction sheet 200 uL were pipette into each well in the MaxiSorpplates. The plates incubated at room temperature and were read at k=562nm on the plate reader.

Data Analysis

The concentrations were calculated for all lysates with the BCA Kit.When lysates are all similar in concentration, then do not normalize forthe MSD analysis. For MSD analysis, average replicate points, calculatestandard deviation, and CV values. Use SigmaPlot to calculate the EC50values for serial dilutions of FGF-21 variants, and use Fold Above WTEC50 as the ranking criteria for the variants. Results can be seen inFIGS. 7a and 7b of this application.

Example 27: FGF-21 Untagged Downstream Process Inclusion Body PrepSolubilization

Cell paste was resuspended by mixing to a final 10% solid in 4° C.inclusion body (IB) Buffer I (50 mM Tris pH 8.0; 100 mM NaCl; 1 mM EDTA;1% Triton X-100; 4° C.). Cells were lysed by passing resuspendedmaterial through a micro fluidizer a total of two times, then it wascentrifuged (10,000 g; 15 min; 4° C.) and the supernatant was decanted.The IB pellet was washed by resuspending in an additional volume of IBbuffer I (50 mM Tris pH 8.0; 100 mM NaCl; 1 mM EDTA; 1% Triton X-100; 4°C.) and resuspended material was passed through micro fluidizer a totalof two times, then it was centrifuged (10,000 g; 15 min; 4° C.) and thesupernatant was decanted. The IB pellet was resuspended in one volume ofbuffer II (50 mM Tris pH 8.0; 100 mM NaCl; 1 mM EDTA; 4° C.), then itwas centrifuged (10,000 g; 15 min; 4° C.) and the supernatant wasdecanted. IB pellet was then resuspended in 1/2 volume of buffer II (50mM Tris pH 8.0; 100 mM NaCl; 1 mM EDTA; 4° C.). IB was aliquoted intoappropriate containers, then it was centrifuged (10,000 g; 15 min; 4°C.) and the supernatant was decanted. Inclusion bodies were solubilized(this is the point at which they could otherwise be stored at −80° C.until further use.)

Inclusion Body Solubilization

Inclusion bodies were solubilized to a final concentration between 10-15mg/mL in solubilization buffer (20 mM Tris, pH 8.0; 8M Urea; 10 mM B-ME)and incubated solubilized IB at room temperature under constant mixingfor 1 hour. Insoluble material was removed by filtration (0.45 μm PESfilter) and the protein concentration was adjusted (not alwaysnecessary) by dilution with additional solubilization buffer (whenprotein concentration is high).

Refold

Refolded by dilution to a final protein concentration of 0.5 mg/mL in 20mM Tris, pH 8.0; 4° C. Allowed to refold for 18 to 24 hours at 4° C.

Purification

Filtered refold reaction through a 0.45 μM PES filter. Loaded materialover a Q HP column (GE Healthcare) equilibrated in Buffer A (20 mM Tris,pH 7.5). Eluted FGF-21 with a linear gradient over 20CV to 100% Buffer B(20mMTris, pH 7.5; 250 mM NaCl). Pooled monomeric FGF-21.

Pegylation and Purification

Took Q HP pool and buffer exchange into 20 mM Tris, pH 8.0; 2M urea; 1mM EDTA. Dropped pH to 4.0 with 50% glacial acetic acid. Concentratesample down to 4.0±1.0 mg/mL. Add 12:1 molar excess PEG and a finalconcentration of 1% Acetic Hydrazide, pH 4.0 to sample. Incubate at 28°C. for 48-72 hours. Add a final of 50 mM Tris base to PEG reaction anddilute 10 fold with RO water. Make sure conductivity is <1 mS/cm and pHis between 8.0-9.0. Load material over a Source 30Q column (GEHealthcare) equilibrated in Buffer A (20 mM Tris, pH 8.0). ElutePEG-FGF-21 with a linear gradient over 20CV to 100% B (20 mM Tris, pH8.0; 100 mM NaCl). Pool PEG-FGF-21 and buffer exchange into 20 mM Tris,pH 7.4; 150 mM NaCl. Concentrate PEG material between 1-2 mg/mL andfilter sterilize using 0.22 μm PES filter. Store at 4° C. For prolongedstorage, flash freeze and store at −80° C.

Example 28

Pharmacokinetic properties of FGF-21 compounds in rats

This protocol was used in order to provide data (found in FIGS. 11-23)on the pharmacokinetic properties of Native and PEG-modified FGF-21compounds produced by Ambrx's proprietary technology in catheterizedrats. The pharmacokinetics of test articles were assayed by ELISAspecific for human FGF-21 from serum samples obtained at specific timepoints after drug dosing.

Test Articles:

1. Ambrx compound PEG-R77 FGF-21 will be used at 0.25 mg/ml diluted in1×PBS.2. Ambrx compound PEG-Y104 FGF-21 will be used at 0.25 mg/ml diluted in1×PBS.3. Ambrx compound PEG-R126 FGF-21 will be used at 0.25 mg/ml diluted in1×PBS.

Test Article Quality/Formulation: Stock Concentrations=

1.0 mg/mL PEG-R77 FGF-211.16 mg/mL PEG-Y104 FGF-211.08 mg/mL PEG-R126 FGF-21

Animals:

Twelve (12) male Sprague-Dawley (SD) rats weighing approximately 250-275grams at study initiation will have had jugular vein catheterssurgically placed prior to arriving at Ambrx. Animals were received fromCRL in good condition and will have acclimated to the study location forat least 3 days prior to the start of the study. Rats will be weighed onthe day of test article administration. Animals will be housed instandard, pathogen-free conditions with food and water ad libitum.

Animal Groups: All compounds will be administered subcutaneouslyGroup 1 (n=4): PEG-R77 SC injection (0.25 mg/kg).Group 2 (n=4): PEG-Y104 SC injection (0.25 mg/kg).Group 3 (n=4): PEG-R126 SC injection (0.25 mg/kg).

Animals are weighed prior to administration of test article. Compoundsare formulated so as to be administered at 1× BW in μL. Subcutaneousadministration of test article is injected into the dorsal scapularregion. Animals will receive a single injection of test article(time=0). At specific time points (see below), whole blood will be drawnfrom the animals, collected into SST microtainer collection tubes. Serumwill be allowed to clot for 30 minutes prior to centrifugation. Serumwill be transferred to polypropylene titer tubes, sealed withmicrostrips, and stored at −800C until analyzed by ELISA to determineFGF-21 serum concentrations.

Data Collection/End point:

Each animal will be used for a complete PK time course. Approximately0.25 mL of whole blood will be drawn from the jugular vein catheters.Immediately after the blood collection, the catheters will be flushedwith 0.1 mL of saline. The following collection time points for animalsreceiving test article material are required based on the anticipatedpharmacokinetic profile of the test articles:

Pre-bleed, 1, 2, 4, 8, 24, 32 48, 56, 72, and 96 hours post-dose.

Termination:

All animals will be euthanized following the completion of the study

Results:

Results are given in the table below and in the figures accompanyingthis application.

FGF21 PEG Isomer PK properties-IV Intravenous PEG 30K 0.25 mg/kg IsomerR72 R77 H87 E91 Y104 E110 R126 P146 Lambda_z 1/hr 0.057 z 0.043 z z0.044 z 0.052 Lambda_z_lower hr 8 z 8 z z 8 z 8 Lambda_z_upper hr 48 z48 z z 48 z 48 HL_Lambda_z hr 12.27 z 16.44 z z 15.61 z 13.67 Tmax hr0.25 z 0.25 z z 0.25 z 0.3125 Cmax ng/mL 5998.6 z 5802.3 z z 7821.4 z8655.8 C0 ng/mL 6861.4 z 6662.5 z z 9280.1 z 9149.9 AUCINF_obs hr*ng/mL53714.9 z 52962.1 z z 69435.8 z 86554.6 Vz_obs mL/kg 82.46 z 113.10 z z81.65 z 58.06 Cl_obs mL/hr/kg 4.65 z 4.74 z z 3.61 z 2.92 MRTINF_obs hr14.49 z 13.81 z z 16.18 z 14.83 Vss_obs mL/kg 67.46 z 65.53 z z 58.53 z43.48

FGF21 PEG Isomer PK properties-SC Subcutaneous PEG 30K 0.25 mg/kg IsomerR72 R77 H87 E91 Y104 E110 R126 P146 Lambda_z 1/hr 0.049 z 0.043 0.035 zz z 0.0317 Lambda_z_lower hr 24 z 24 24 z z z 24 Lambda_z_upper hr 96 z90 96 z z z 96 HL_Lambda_z hr 14.72 z 16.14 19.93 z z z 22.01 Tmax hr 24z 24 22 z z z 24 Cmax ng/mL 254.5 z 174.3 229.7 z z z 321.3 AUCINF_obshr*ng/mL 11824.7 z 8206.7 12177.2 z z z 15908.4 Vz_obs mL/kg 458.9 z731.1 606.2 z z z 503.4 Cl_obs mL/hr/kg 21.92 z 31.67 20.91 z z z 15.86MRTINF_obs hr 36.52 z 36.31 40.35 z z z 40.07Increased Tmax of PEGylated compoundsIncreased T_(1/2) of PEGylated compoundsIncreased AUC of PEGylated compoundsPEGylation site-dependent PK attributes

Example 29

In Vivo Studies of PEGylated FGF-21

PEG-FGF-21, unmodified FGF-21 and buffer solution are administered tomice or rats. The results will show superior activity and prolonged halflife of the PEGylated FGF-21 of the present invention compared tounmodified FGF-21. Similarly, modified FGF-21, unmodified FGF-21, andbuffer solution are administered to mice or rats.

Pharmacokinetic Analysis

WO 2005/091944 describes pharmacokinetic studies that can be performedwith the FGF-21 compounds of the present invention. A FGF-21 polypeptideof the invention is administered by intravenous or subcutaneous routesto mice. The animals are bled prior to and at time points after dosing.Plasma is collected from each sample and analyzed by radioimmunoassay.Elimination half-life can be calculated and compared between FGF-21polypeptides comprising a non-naturally encoded amino acid and wild-typeFGF-21 or various forms of FGF-21 polypeptides of the invention.Similarly, FGF-21 polypeptides of the invention may be administered tocynomolgus monkeys. The animals are bled prior to and at time pointsafter dosing. Plasma is collected from each sample and analyzed byradioimmunoassay.

Polypeptides of the invention may be administered to ZDF male rats(diabetic, fat rats; 8 weeks of age at beginning of study, CharlesRiver-GMI). Rats are fed Purina 5008 feed ad libitum. The following testgroups are set up: Saline; Insulin 4U/day; FGF-21, 500 ug/day Acute(Acute dosing group is dosed once and bled at T=0, 2, 4, 8, and 24 hourspost dose); FGF-21, 100 ug/day; FGF-21, 250 ug/day; FGF-21, 500 ug/day;FGF-21(once/day) 500 ug/ml; Lean Saline; Lean Insulin 4U/day; LeanFGF-21 500 ug/day. Lean groups represent non-diabetic, lean, ZDF rats.

Compounds are injected s.c. (b.i.d.), except for the second 500 ug/daygroup which receives one injection per day for the duration of the study(7 days). Control rats are injected with vehicle (PBS; 0.1 ml). Following 7 days of dosing, the animals are subjected to an oral glucosetolerance test. Blood for glucose and triglycerides are collected bytail clip bleeding without anesthetic. FGF-21 polypeptides may reduceplasma glucose levels in a dose-dependent manner. Also lean lean ZDFrats may not become hypoglycemic after exposure to FGF-21 polypeptidesof the invention when compared to rats dosed with insulin.

ob/ob Obesity Model

The ob/ob mouse model is an animal model for hyperglycemia, insulinresistance, and obesity. Plasma glucose levels after treatment withFGF-21 polypeptide compared to vehicle and insulin control groups may bemeasured in ob/ob mice. In this obesity model, the test groups of maleob/ob mice (7 weeks old) are injected with vehicle alone (PBS), insulin(4 U/day), or FGF-21 polypeptide (5 μg/day and 25 μg/day),subcutaneously (0.1 ml, b.i.d) for seven days. Blood is collected bytail clip bleeding on days 1, 3, and 7, one hour after the firstcompound injection, and plasma glucose levels are measured using astandard protocol. FGF-21 polypeptides of the invention stimulateglucose uptake if they reduce plasma glucose levels when compared to thevehicle control group. Triglyceride levels may be compared aftertreatment with FGF-21 polypeptides of the invention compared to othermolecules. The polypeptide may be administered the mice via multipledoses, continuous infusion, or a single dose, etc.

Example 30

Pharmacokinetic evaluation of FGF21 analogs: The pharmacokineticproperties of 30KPEG-pAF(N₆-His)FGF21 analogs with varying sites of PEGconjugation were evaluated in rat. Other parameters studied were PEG MW,as well as dose of compound administered. The percent bioavailabilityfor a few 30KPEG-pAF(N₆-His)FGF21 variants was determined.

Animals: All animal experimentation was conducted under protocolsapproved by the Institutional Animal Care and Use Committee. Male(175-300 g) Sprague-Dawley rats were obtained from Charles RiverLaboratories. Rats were housed individually in cages in rooms with a12-h light/dark cycle and acclimated to the Ambrx vivarium for at least3 days prior to experimentation. Animals were provided access tocertified Purina rodent chow 5001 and water ad libitum.

Dosing and Serum Collection: Catheters were surgically installed intothe jugular vein for blood collection by CRL prior to shipment.Following successful catheter patency, animals were assigned totreatment groups prior to dosing. A single-dose of compound wasadministered intravenously or subcutaneously in a dose volume of 1mL/kg. Compound dose concentrations were derived by dilution in PBSusing the stock concentration as assigned in the Certificate of Release.Blood samples were collected at various time points via the indwellingcatheter and placed into SST microfuge tubes. Serum was collected aftercentrifugation, and stored at −80° C. until analysis.

Pharmacokinetics Analysis: The assay for the quantification ofPEG-FGF-21 in Sprague-Dawley rat serum was developed at Ambrx Inc., LaJolla, Calif. Microplate wells are coated with goat anti-human FGF-21IgG polyclonal antibody (PAb; RnD Systems, clone AF2539) that is used asthe capture reagent. Standard (STD) and quality control (QC) samples,both made by spiking PEG-FGF-21 analog into 100% Sprague Dawley ratserum, and study samples are loaded into the wells after pre-treating1:100 with I-Block buffer. The FGF-21 in the STDs, QCs and study samplesis captured by the immobilized PAb. Unbound materials are removed bywashing the wells. Biotin goat anti-human FGF-21 IgG PAb (RnD Systems,clone BAF2539) is added to the wells followed by a wash step and theaddition of streptavidin horseradish peroxidase (SA-HRP; RnD Systems,Catalog # DY998) for detection of the captured PEG-FGF-21. After anotherwashing step, tetramethylbenzidine (TMB, Kirkegaard Perry Laboratories)substrate solution is added to the wells. TMB reacts with the peroxidein the presence of HRP and produces a colorimetric signal proportionalto the amount of PEG-FGF-21 analog bound by the capture reagent in theinitial step. The color development is stopped by the addition of 2Nsulphuric acid and the intensity of the color (optical density, OD) ismeasured at 450 nm. The conversion of OD units for the study samples andthe QCs to concentration is achieved through a computer softwaremediated comparison to a standard curve on the same plate, which isregressed according to a 5-parameter logistic regression model usingSOFTmax Pro v5 data reduction package. Results are reported in ng/mLconcentration units.

Concentrations may also be measured by a double antibody sandwich assayor other methods known to those skilled in the art. Concentrations werecalculated using a standard curve generated from the corresponding dosedcompound. Pharmacokinetic parameters were estimated using the modelingprogram WinNonlin (Pharsight, version 4.1). Noncompartmental analysisfor individual animal data with linear-up/log-down trapezoidalintegration was used, and concentration data was uniformly weighted.

Conclusions: The pharmacokinetic properties of WT N₆-His FGF21 was inline with that reported by Kharitonenkov et al, 2005 and was comparableto the non-tagged WT FGF21 protein.

The pharmacokinetic profiles of the 30KPEG-pAF(N₆-His)FGF21 isomers weresignificantly increased in by the addition of a 30 kDa PEG molecule ascompared to results obtained from WT (un-PEGylated) FGF21.

The PEGylated compounds exhibited markedly different PK profiles whendosed at the 0.25 mg/kg level subcutaneously. H87 and L86 tended to haveinferior PK attribute as compared to the other isomers. Further, R131and Q108 PEG30 compounds differentially generated superior PKproperties. More specifically, these compound had improved AUC, Cmax andterminal half-life. An in-depth structure-activity analysis may revealstructural explanations for the various PK properties of each isomer.

Comparison of PEG molecular weight showed 30KPEG-pAF91(N₆-His)FGF21 tohave a slightly greater persistence in the circulation than20KPEG-pAF91(N₆-His)FGF21. However, as the 20 kDa isomer had a higherCmax value, the total AUCinf for the two compounds was comparable.Bioavailability for the 20 kDa isoform was slightly better than the 30kDa variant at 30% versus 20%, respectively.

TABLE 3 Pharmacokinetic parameter values for compounds dosed 0.25 mg/kgsubcutaneously in rat. 0.25 mg/kg Terminal t_(1/2) Tmax Cmax AUC_(all)AUC_(INF) Vz/f Cl/f MRT_(INF) SC hr hr ng/mL hr*ng/mL hr*ng/mL mL/kgmL/hr/kg hr N6His WT 1.26 0.9 92.1 261.6 297.3 1768.9 971.2 2.52 PP WT1.19 1.5 96.6 259.8 294.7 1576.2 910.4 2.53 R72 14.72 24 254.5 NE11824.7 458.9 21.9 36.52 R77 32.30 22 237.0 14571.9 17687.8 655.8 14.459.18 L86 33.90 27 52.2 3215.7 3928.8 3153.3 66.0 59.27 H87 16.14 24174.3 NE 8206.7 731.1 31.7 36.31 E91 19.93 22 229.7 NE 12177.2 606.220.9 40.35 Y104 12.37 24 321.9 19085.8 21188.1 416.3 11.9 47.63 Q10827.31 24 590.3 31837.4 36387.1 272.4 7.0 51.71 R126 16.48 20 248.113238.3 15033.4 643.0 16.8 49.57 R131 26.13 24 545.4 27373.4 30786.0315.0 8.3 50.41 P146 22.01 24 321.3 NE 15908.4 503.4 15.9 40.07

Serum concentration versus time curves were evaluated bynoncompartmental analysis (Pharsight, version 4.1). N═3-4 rats percompound. ND: not done; NE: not evaluated. Tmax: time to reach Cmax;Cmax: maximum concentration; terminal tv2: terminal half-life;AUC_(last): area under the concentration-time curve to the last plasmasample/timepoint; AUC_(inf): area under the concentration-time curveextrapolated to infinity; MRT: mean residence time; Cl/f: apparent totalplasma clearance; Vz/f: apparent volume of distribution during terminalphase.

TABLE 4 Pharmacokinetic parameter values for 20KPEG-pAF91(N6-hHis) FGF21dosed 0.25 mg/kg subcutaneously in rat. Rat #1 Rat #2 Rat #3 Rat #4Terminal t_(1/2) hr 22.6 24.4 17.3 16.9 Tmax hr 8 8 8 24 Cmax ng/mL163.0 182.1 155.7 88.8 AUC_(all) hr*ng/mL 7629.1 7659.1 6461.4 4375.4AUC_(INF) hr*ng/mL 8232.0 8333.8 6661.2 4521.7 Vz/f mL/kg 989.8 1054.2937.3 1347.7 Cl/f mL/hr/kg 30.4 30.0 37.5 55.3 MRT_(INF) hr 39.9 41.032.5 34.3Concentration versus time curves were evaluated by noncompartmentalanalysis (Pharsight, version 4.1). ND: not done; NE: could not beevaluated. Tmax: time to reach Cmax; Cmax: maximum concentration;terminal t_(1/2): terminal half-life; AUC_(last): area under theconcentration-time curve to the last plasma sample/timepoint; AUC_(inf):area under the concentration-time curve extrapolated to infinity; MRT:mean residence time; Cl/f: apparent total plasma clearance; Vz/f:apparent volume of distribution during terminal phase.

Example 31

Human Clinical Trial of the Safety and/or Efficacy of PEGylated FGF-21Comprising a Non-Naturally Encoded Amino Acid.

Objective To observe the safety and pharmacokinetics of subcutaneouslyadministered PEGylated recombinant human FGF-21 comprising anon-naturally encoded amino acid.

Patients Eighteen healthy volunteers ranging between 20-40 years of ageand weighing between 60-90 kg are enrolled in the study. The subjectswill have no clinically significant abnormal laboratory values forhematology or serum chemistry, and a negative urine toxicology screen,HIV screen, and hepatitis B surface antigen. They should not have anyevidence of the following: hypertension; a history of any primaryhematologic disease; history of significant hepatic, renal,cardiovascular, gastrointestinal, genitourinary, metabolic, neurologicdisease; a history of anemia or seizure disorder; a known sensitivity tobacterial or mammalian-derived products, PEG, or human serum albumin;habitual and heavy consumer to beverages containing caffeine;participation in any other clinical trial or had blood transfused ordonated within 30 days of study entry; had exposure to FGF-21 withinthree months of study entry; had an illness within seven days of studyentry; and have significant abnormalities on the pre-study physicalexamination or the clinical laboratory evaluations within 14 days ofstudy entry. All subjects are evaluable for safety and all bloodcollections for pharmacokinetic analysis are collected as scheduled. Allstudies are performed with institutional ethics committee approval andpatient consent.

Study Design This will be a Phase I, single-center, open-label,randomized, two-period crossover study in healthy male volunteers.Eighteen subjects are randomly assigned to one of two treatment sequencegroups (nine subjects/group). FGF-21 is administered over two separatedosing periods as a bolus s.c. injection in the upper thigh usingequivalent doses of the PEGylated FGF-21 comprising a non-naturallyencoded amino acid and the commercially available product chosen. Thedose and frequency of administration of the commercially availableproduct is as instructed in the package label. Additional dosing, dosingfrequency, or other parameter as desired, using the commerciallyavailable products may be added to the study by including additionalgroups of subjects. Each dosing period is separated by a 14-day washoutperiod. Subjects are confined to the study center at least 12 hoursprior to and 72 hours following dosing for each of the two dosingperiods, but not between dosing periods. Additional groups of subjectsmay be added if there are to be additional dosing, frequency, or otherparameter, to be tested for the PEGylated FGF-21 as well. Theexperimental formulation of FGF-21 is the PEGylated FGF-21 comprising anon-naturally encoded amino acid.

Blood Sampling Serial blood is drawn by direct vein puncture before andafter administration of FGF-21. Venous blood samples (5 mL) fordetermination of serum FGF-21 concentrations are obtained at about 30,20, and 10 minutes prior to dosing (3 baseline samples) and atapproximately the following times after dosing: 30 minutes and at 1, 2,5, 8, 12, 15, 18, 24, 30, 36, 48, 60 and 72 hours. Each serum sample isdivided into two aliquots. All serum samples are stored at −20° C. Serumsamples are shipped on dry ice. Fasting clinical laboratory tests(hematology, serum chemistry, and urinalysis) are performed immediatelyprior to the initial dose on day 1, the morning of day 4, immediatelyprior to dosing on day 16, and the morning of day 19.

Bioanalytical Methods An ELISA kit is used for the determination ofserum FGF-21 concentrations.

Safety Determinations Vital signs are recorded immediately prior to eachdosing (Days 1 and 16), and at 6, 24, 48, and 72 hours after eachdosing. Safety determinations are based on the incidence and type ofadverse events and the changes in clinical laboratory tests frombaseline. In addition, changes from pre-study in vital signmeasurements, including blood pressure, and physical examination resultsare evaluated.

Data Analysis Post-dose serum concentration values are corrected forpre-dose baseline FGF-21 concentrations by subtracting from each of thepost-dose values the mean baseline FGF-21 concentration determined fromaveraging the FGF-21 levels from the three samples collected at 30, 20,and 10 minutes before dosing. Pre-dose serum FGF-21 concentrations arenot included in the calculation of the mean value if they are below thequantification level of the assay. Pharmacokinetic parameters aredetermined from serum concentration data corrected for baseline FGF-21concentrations. Pharmacokinetic parameters are calculated by modelindependent methods on a Digital Equipment Corporation VAX 8600 computersystem using the latest version of the BIOAVL software. The followingpharmacokinetics parameters are determined: peak serum concentration(Cmax); time to peak serum concentration (tmax); area under theconcentration-time curve (AUC) from time zero to the last blood samplingtime (AUCo-72) calculated with the use of the linear trapezoidal rule;and terminal elimination half-life (tv2), computed from the eliminationrate constant. The elimination rate constant is estimated by linearregression of consecutive data points in the terminal linear region ofthe log-linear concentration-time plot. The mean, standard deviation(SD), and coefficient of variation (CV) of the pharmacokineticparameters are calculated for each treatment. The ratio of the parametermeans (preserved formulation/non-preserved formulation) is calculated.

Safety Results The incidence of adverse events is equally distributedacross the treatment groups. There are no clinically significant changesfrom baseline or pre-study clinical laboratory tests or blood pressures,and no notable changes from pre-study in physical examination resultsand vital sign measurements. The safety profiles for the two treatmentgroups should appear similar.

Pharmacokinetic Results Mean serum FGF-21 concentration-time profiles(uncorrected for baseline FGF-21 levels) in all 18 subjects afterreceiving PEGylated FGF-21 comprising a non-naturally encoded amino acidat each time point measured. All subjects should have pre-dose baselineFGF-21 concentrations within the normal physiologic range.Pharmacokinetic parameters are determined from serum data corrected forpre-dose mean baseline FGF-21 concentrations and the Cmax and tmax aredetermined. The mean tmax for the any clinical comparator(s) chosen issignificantly shorter than the tmax for the PEGylated FGF-21 comprisingthe non-naturally encoded amino acid. Terminal half-life values aresignificantly shorter for the preclinical comparator(s) tested comparedwith the terminal half-life for the PEGylated FGF-21 comprising anon-naturally encoded amino acid.

Although the present study is conducted in healthy male subjects,similar absorption characteristics and safety profiles would beanticipated in other patient populations; such as male or femalepatients with cancer or chronic renal failure, pediatric renal failurepatients, patients in autologous predeposit programs, or patientsscheduled for elective surgery.

In conclusion, subcutaneously administered single doses of PEGylatedFGF-21 comprising non-naturally encoded amino acid will be safe and welltolerated by healthy male subjects. Based on a comparative incidence ofadverse events, clinical laboratory values, vital signs, and physicalexamination results, the safety profiles of the commercially availableforms of FGF-21 and PEGylated FGF-21 comprising non-naturally encodedamino acid will be equivalent. The PEGylated FGF-21 comprisingnon-naturally encoded amino acid potentially provides large clinicalutility to patients and health care providers.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to those of ordinary skill in the artand are to be included within the spirit and purview of this applicationand scope of the appended claims. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

TABLE 5 Sequences Cited. SEQ ID # Sequence Name  1Amino acid sequence of FGF-21 without leader (P form)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS  2Amino acid sequence of FGF-21 without leader (P form)-His taggedMHHHHHHSGGHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS  3Amino acid sequence of FGF-21 with leader (P form)-leader with 3 leucines(209 amino acid P-form)MDSDETGFEHSGLWVSVLAGLLLGACQAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQ GRSPSYAS  4Amino acid sequence of FGF-21 with leader (P form)-leader with two leucinesMDSDETGFEHSGLWVSVLAGLLGACQAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQG RSPSYAS  5Amino acid sequence of FGF-21 without leader (L form)His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln ArgTyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu AspGly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys AlaLeu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys GlnArg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His The Asp Pro Glu Ala Cys Ser PheArg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His GlyLeu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg GlyPro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Len Pro Glu Pro Pro GlyIle Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val GlyPro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser  6Amino acid sequence of FGF-21 with leader (L form)-leader with 3 leucines(209 amino acid L-form)Met Asp Ser Asp Glu Thr Gly Phe Glu His Ser Gly Leu Trp Val Ser Val Leu AlaGly Leu Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro Asp Ser Ser Pro Leu LeuGln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln GlnThr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp GlnSer Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile LeuGly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly SerLeu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp GlyTyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Asn LysSer Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro GlyLeu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp ValGly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser TyrAla Ser  7Amino acid sequence of FGF-21 with leader (L form)-leader with 2 leucines(208 amino acid L-form)Met Asp Ser Asp Glu Thr Gly Phe Glu His Ser Gly Leu Trp Val Ser Val Leu AlaGly Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro Asp Ser Ser Pro Leu Leu GlnPhe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln ThrGlu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln SerPro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu GlyVal Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser LeuHis Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly TyrAsn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Asn Lys SerPro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly LeuPro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val GlySer Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr AlaSer  8 Nucleotide Sequence for FGF-21 without leader (P form)CACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTCGGGGGCCAAGTCCGGCAGCGGTACCTCTACACAGATGATGCCCAGCAGACAGAAGCCCACCTGGAGATCAGGGAGGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTCCTGCAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGACATCCAGGTTCCTGTGCCAGCGGCCAGATGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCACCTGCCAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGGACCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCCGCACCCCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCCCCCGATGTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCCCAGGGCCGA AGCCCCAGCTACGCTTCCTGA 9 Nucleotide Sequence for FGF-21 without leader (P form)-His taggedATGCATCATCATCATCATCATAGCGGCGGCCACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTCGGGGGCCAAGTCCGGCAGCGGTACCTCTACACAGATGATGCCCAGCAGACAGAAGCCCACCTGGAGATCAGGGAGGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTCCTGCAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGACATCCAGGTTCCTGTGCCAGCGGCCAGATGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCACCTGCCAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGGACCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCCGCACCCCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCCCCCGATGTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCCCAGGGCCGAAGCCCCAGCTACGCTTC CTGA 10Nucleotide Sequence for FGF-21 with leader (P form)-leader with 3 leucinesATGGACTCGGACGAGACCGGGTTCGAGCACTCAGGACTGTGGGTTTCTGTGCTGGCTGGTCTTCTGCTGGGAGCCTGCCAGGCACACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTCGGGGGCCAAGTCCGGCAGCGGTACCTCTACACAGATGATGCCCAGCAGACAGAAGCCCACCTGGAGATCAGGGAGGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTCCTGCAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGACATCCAGGTTCCTGTGCCAGCGGCCAGATGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCACCTGCCAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGGACCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCCGCACCCCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCCCCCGATGTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCCCAGGGCCGAAGCCCCAGCTAC GCTTCCTGA 11Nucleotide Sequence for FGF-21 with leader (P form)-leader with 2 leucinesATGGACTCGGACGAGACCGGGTTCGAGCACTCAGGACTGTGGGTTTCTGTGCTGGCTGGTCTTCTGGGAGCCTGCCAGGCACACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTCGGGGGCCAAGTCCGGCAGCGGTACCTCTACACAGATGATGCCCAGCAGACAGAAGCCCACCTGGAGATCAGGGAGGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTCCTGCAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGACATCCAGGTTCCTGTGCCAGCGGCCAGATGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCACCTGCCAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGGACCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCCGCACCCCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCCCCCGATGTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCCCAGGGCCGAAGCCCCAGCTACGCT TCCTGA 12Nucleotide Sequence for FGF-21 without leader (L form)CACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTCGGGGCCAAGTCCGGCAGCGGTACCTCTACACAGATGATGCCCAGCAGACAGAAGCCCACCTGGAGATCAGGGAGGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTCCTGCAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGACATCCAGGTTCCTGTGCCAGCGGCCAGATGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCACCTGCCAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGGACCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCCGCACTCCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCCCCCGATGTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCCCAGGGCCGAAG CCCAGCTACGCTTCCTGA 13Nucleotide Sequence for FGF-21 with leader (L form)-leader with 3 leucinesATG GAC TCG GAC GAG ACC GGG TTC GAG CAC TCA GGA CTG TGGGTT TCT GTG CTG GCT GGT CTT CTG CTG GGA GCC TGC CAG GCACAC CCC ATC CCT GAC TCC AGT CCT CTC CTG CAA TTC GGG GGCCAA GTC CGG CAG CGG TAC CTC TAC ACA GAT GAT GCC CAG CAGACA GAA GCC CAC CTG GAG ATC AGG GAG GAT GGG ACG GTG GGGGGC GCT GCT GAC CAG AGC CCC GAA AGT CTC CTG CAG CTG AAAGCC TTG AAG CCG GGA GTT ATT CAA ATC TTG GGA GTC AAG ACATCC AGG TTC CTG TGC CAG CGG CCA GAT GGG GCC CTG TAT GGATCG CTC CAC TTT GAC CCT GAG GCC TGC AGC TTC CGG GAG CTGCTT CTT GAG GAC GGA TAC AAT GTT TAC CAG TCC GAA GCC CACGGC CTC CCG CTG CAC CTG CCA GGG AAC AAG TCC CCA CAC CGGGAC CCT GCA CCC CGA GGA CCA GCT CGC TTC CTG CCA CTA CCAGGC CTG CCC CCC GCA CTC CCG GAG CCA CCC GGA ATC CTG GCCCCC CAG CCC CCC GAT GTG GGC TCC TCG GAC CCT CTG AGC ATGGTG GGA CCT TCC CAG GGC CGA AGC CCC AGC TAC GCT TCC TGA 14Nucleotide Sequence for FGF-21 with leader (L form)-leader with 2 leucinesATG GAC TCG GAC GAG ACC GGG TTC GAG CAC TCA GGA CTG TGGGTT TCT GTG CTG GCT GGT CTT CTG GGA GCC TGC CAG GCA CACCCC ATC CCT GAC TCC AGT CCT CTC CTG CAA TTC GGG GGC CAAGTC CGG CAG CGG TAC CTC TAC ACA GAT GAT GCC CAG CAG ACAGAA GCC CAC CTG GAG ATC AGG GAG GAT GGG ACG GTG GGG GGCGCT GCT GAC CAG AGC CCC GAA AGT CTC CTG CAG CTG AAA GCCTTG AAG CCG GGA GTT ATT CAA ATC TTG GGA GTC AAG ACA TCCAGG TTC CTG TGC CAG CGG CCA GAT GGG GCC CTG TAT GGA TCGCTC CAC TTT GAC CCT GAG GCC TGC AGC TTC CGG GAG CTG CTTCTT GAG GAC GGA TAC AAT GTT TAC CAG TCC GAA GCC CAC GGCCTC CCG CTG CAC CTG CCA GGG AAC AAG TCC CCA CAC CGG GACCCT GCA CCC CGA GGA CCA GCT CGC TTC CTG CCA CTA CCA GGCCTG CCC CCC GCA CTC CCG GAG CCA CCC GGA ATC CTG GCC CCCCAG CCC CCC GAT GTG GGC TCC TCG GAC CCT CTG AGC ATG GTGGGA CCT TCC CAG GGC CGA AGC CCC AGC TAC GCT TCC TGA 34Amino acid sequence of FGF-21 (Rattus norvegicus-ref|NP_570108.1|[18543365])Met Asp Trp Met Lys Ser Arg Val Gly Ala Pro Gly Leu Trp Val Cys Leu LeuLeu Pro Val Phe Leu Leu Gly Val Cys Glu Ala Tyr Pro Ile Ser Asp Ser Ser ProLeu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp AspGln Asp Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Val Gly Thr AlaHis Arg Ser Pro Glu Ser Leu Leu Glu Leu Lys Ala Leu Lys Pro Gly Val Ile GlnIle Leu Gly Val Lys Ala Ser Arg Phe Leu Cys Gln Gln Pro Asp Gly Thr Leu TyrGly Ser Pro His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Lys AspGly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu Arg Leu Pro Gln LysAsp Ser Gln Asp Pro Ala Thr Arg Gly Pro Val Arg Phe Leu Pro Met Pro Gly LeuPro His Glu Pro Gln Glu Gln Pro Gly Val Leu Pro Pro Glu Pro Pro Asp Val GlySer Ser Asp Pro Leu Ser Met Val Glu Pro Leu Gln Gly Arg Ser Pro Ser Tyr AlaSer 35Amino acid sequence of FGF-21 (Mus musculus-ref|NP_064397.1|[9910218])Met Glu Trp Met Arg Ser Arg Val Gly Thr Leu Gly Leu Trp Val Arg Leu LeuLeu Ala Val Phe Leu Leu Gly Val Tyr Gln Ala Tyr Pro Ile Pro Asp Ser Ser ProLeu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp AspGln Asp Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Val Gly Ala AlaHis Arg Ser Pro Glu Ser Leu Leu Glu Leu Lys Ala Leu Lys Pro Gly Val Ile GlnIle Leu Gly Val Lys Ala Ser Arg Phe Leu Cys Gln Gln Pro Asp Gly Ala Leu TyrGly Ser Pro His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu AspGly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu Arg Leu Pro Gln LysAsp Ser Pro Asn Gln Asp Ala Thr Ser Trp Gly Pro Val Arg Phe Leu Pro Met ProGly Leu Leu His Glu Pro Gln Asp Gln Ala Gly Phe Leu Pro Pro Glu Pro Pro AspVal Gly Ser Ser Asp Pro Leu Ser Met Val Glu Pro Leu Gln Gly Arg Ser Pro SerTyr Ala Ser 36Amino acid sequence of FGF-21 (Danio rerio-ref|NP_001038789.1|[113671792])Met Leu Phe Ala Cys Phe Phe Ile Phe Phe Ala Leu Phe Pro His Leu Arg Trp CysMet Tyr Val Pro Ala Gln Asn Val Leu Leu Gln Phe Gly Thr Gln Val Arg GluArg Leu Leu Tyr Thr Asp Gly Leu Phe Leu Glu Met Asn Pro Asp Gly Ser ValLys Gly Ser Pro Glu Lys Asn Leu Asn Cys Val Leu Glu Leu Arg Ser Val Lys AlaGly Glu Thr Val Ile Gln Ser Ala Ala Thr Ser Leu Tyr Leu Cys Val Asp Asp GlnAsp Lys Leu Lys Gly Gln His His Tyr Ser Ala Leu Asp Cys Thr Phe Gln Glu LeuLeu Leu Asp Gly Tyr Ser Phe Phe Leu Ser Pro His Thr Asn Leu Pro Val Ser LeuLeu Ser Lys Arg Gln Lys His Gly Asn Pro Leu Ser Arg Phe Leu Pro Val Ser ArgAla Glu Asp Ser Arg Thr Gln Glu Val Lys Gln Tyr Ile Gln Asp Ile Asn Leu AspSer Asp Asp Pro Leu Gly Met Gly His Arg Ser His Leu Gln Thr Val Phe Ser ProSer Leu His Thr Lys Lys 37Amino acid sequence of Klotho beta (Homo sapiens-ref|NP_783864.1|[28376633])MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAVTGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSWKKDGKGPSIWDHFIHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQFSISWPRLFPDGIVTVANAKGLQYYSTLLDALVLRNIEIVTLYHWDLPLALQEKYGGWKNDTIIDIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGYGTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITLGSHWIEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFSVLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREALNWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLDEIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYKQIIRENGFSLKESTPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPHLYVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHAHLGLPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDIYNRSGNDTYGAAHNLLVAHALAWRLYDRQFRPSQRGAVSLSLHADWAEPANPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFKAKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFLGCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRVVS 38Amino acid sequence of Klotho beta (Mus musculus-refNP_112457.1GI:13626032) MKTGCAAGSPGNEWIFFSSDERNTRSRKTMSNRALQRSAVLSAFVLLRAVTGFSGDGKAIWDKKQYVSPVNPSQLFLYDTFPKNFSWGVGTGAFQVEGSWKTDGRGPSIWDRYVYSHLRGVNGTDRSTDSYIFLEKDLLALDFLGVSFYQFSISWPRLFPNGTVAAVNAQGLRYYRALLDSLVLRNIEPIVTLYHWDLPLTLQEEYGGWKNATMIDLFNDYATYCFQTFGDRVKYWITIHNPYLVAWHGFGTGMHAPGEKGNLTAVYTVGHNLIKAHSKVWHNYDKNFRPHQKGWLSITLGSHWIEPNRTDNMEDVINCQHSMSSVLGWFANPIHGDGDYPEFMKTGAMIPEFSEAEKEEVRGTADFFAFSFGPNNFRPSNTVVKMGQNVSLNLRQVLNWIKLEYDDPQILISENGWFTDSYIKTEDTTAIYMMKNFLNQVLQAIKFDEIRVFGYTAWTLLDGFEWQDAYTTRRGLFYVDFNSEQKERKPKSSAHYYKQIIQDNGFPLKESTPDMKGRFPCDFSWGVTESVLKPEFTVSSPQFTDPHLYVWNVTGNRLLYRVEGVRLKTRPSQCTDYVSIKKRVEMLAKMKVTHYQFALDWTSILPTGNLSKVNRQVLRYYRCVVSEGLKLGVFPMVTLYHPTHSHLGLPLPLLSSGGWLNMNTAKAFQDYAELCFRELGDLVKLWITINEPNRLSDMYNRTSNDTYRAAHNLMIAHAQVWHLYDRQYRPVQHGAVSLSLHCDWAEPANPFVDSHWKAAERFLQFEIAWFADPLFKTGDYPSVMKEYIASKNQRGLSSSVLPRFTAKESRLVKGTVDFYALNHFTTRFVIHKQLNTNRSVADRDVQFLQDITRLSSPSRLAVTPWGVRKLLAWIRRNYRDRDIYITANGIDDLALEDDQIRKYYLEKYVQEALKAYLIDKVKIKGYYAFKLTEEKSKPRFGFFTSDFRAKSSVQFYSKLISSSGLPAENRSPACGQPAEDTDCTICSFLVEKKPLIFFGCCFISTLAVLLSITVFHHQKRRKFQKARNLQ NIPLKKGHSRVFS 39OmpA nucleotide leader sequenceatgaaaaaaactgctatcgcgatcgctgtagctctggctggtttcgcgaccgtagctaacgct 40OmpA amino acid leader sequenceM K K T A I A I A V A L A G F A T V A N A 41MalE nucleotide leader sequenceatgaaaataaaaacaggtgcacgcatcctcgcattatccgcattaacgacgatgatgttttccgccteggctctcgcc42 MalE amino acid leader sequenceM K I K T G A R I L A L S A L T T M M F S A S A L A 43StII nucleotide leader sequenceatgaaaaagaatatcgcatttatatgcatctatgttcgttttttctattgctacaaatgcctatgca 44StII amino acid leader sequenceM K K N I A F L L A S M F V F S I A T N A Y A

1. A FGF-21 polypeptide comprising one or more non-naturally encodedamino acids.
 2. (canceled)
 3. The FGF-21 polypeptide of claim 1, whereinthe polypeptide is linked to a linker, polymer, or biologically activemolecule.
 4. The FGF-21 polypeptide of claim 3, wherein the polypeptideis linked to a water soluble polymer. 5-7. (canceled)
 8. The FGF-21polypeptide of claim 4, wherein the water soluble polymer comprises apoly(ethylene glycol) moiety.
 9. The FGF-21 polypeptide of claim 4,wherein said water soluble polymer is linked to a non-naturally encodedamino acid present in said FGF-21 polypeptide.
 10. (canceled)
 11. TheFGF-21 polypeptide of claim 1, wherein the non-naturally encoded aminoacid is substituted at a position selected from the group consisting ofresidues 10, 52, 117, 126, 131, 162, 87, 77, 83, 72, 69, 79, 91, 96,108, 110, and any combination thereof (SEQ ID NO: 1 or the correspondingamino acids in SEQ ID NOs: 2-7). 12-26. (canceled)
 27. The FGF-21polypeptide of claim 1, wherein the non-naturally encoded amino acidcomprises a carbonyl group, an aminooxy group, a hydrazine group, ahydrazide group, a semicarbazide group, an azide group, or an alkynegroup. 28-37. (canceled)
 38. The FGF-21 polypeptide of claim 4, whereinthe water soluble polymer has a molecular weight of between about 0.1kDa and about 100 kDa.
 39. (canceled)
 40. The FGF-21 polypeptide ofclaim 4, which is made by reacting a FGF-21 polypeptide comprising acarbonyl-containing amino acid with a water soluble polymer comprisingan aminooxy, hydrazine, hydrazide or semicarbazide group.
 41. The FGF-21polypeptide of claim 40, wherein the aminooxy, hydrazine, hydrazide orsemicarbazide group is linked to the water soluble polymer through anamide linkage.
 42. The FGF-21 polypeptide of claim 4, which is made byreacting a water soluble polymer comprising a carbonyl group with apolypeptide comprising a non-naturally encoded amino acid that comprisesan aminooxy, a hydrazine, a hydrazide or a semicarbazide group. 43-53.(canceled)
 54. An isolated nucleic acid that encodes a modified FGF-21polypeptide according to claim 1, wherein the polynucleotide comprisesat least one selector codon that encodes said one or more non-naturallyencoded amino acids.
 55. The isolated nucleic acid of claim 54, whereinthe selector codon is selected from the group consisting of an ambercodon, ochre codon, opal codon, a unique codon, a rare codon, and afour-base codon.
 56. A method of making the FGF-21 polypeptide of claim3, the method comprising contacting an isolated FGF-21 polypeptidecomprising a non-naturally encoded amino acid with a linker, polymer, orbiologically active molecule comprising a moiety that reacts with thenon-naturally encoded amino acid. 57-65. (canceled)
 66. A compositioncomprising the FGF-21 polypeptide of claim 1 and a pharmaceuticallyacceptable carrier.
 67. (canceled)
 68. A method of treating a patienthaving a disorder modulated by FGF-21 comprising administering to thepatient a therapeutically-effective amount of the composition of claim66.
 69. A cell comprising the nucleic acid of claim
 54. 70. (canceled)71. A method of making a modified FGF-21 polypeptide according to claim1, the method comprising, culturing cells comprising a polynucleotide orpolynucleotides encoding a FGF-21 polypeptide comprising a selectorcodon, an orthogonal RNA synthetase and an orthogonal tRNA underconditions to permit expression of the FGF-21 polypeptide comprising anon-naturally encoded amino acid; and purifying the FGF-21 polypeptide.72. A method of modulating serum half-life or circulation time of aFGF-21 polypeptide, the method comprising substituting one or morenon-naturally encoded amino acids for any one or more naturallyoccurring amino acids in the FGF-21 polypeptide. 73-97. (canceled)
 98. Amethod of modulating immunogenicity of a FGF-21 polypeptide, the methodcomprising substituting one or more non-naturally encoded amino acidsfor any one or more naturally occurring amino acids in the FGF-21polypeptide. 99-111. (canceled)